Abstract:

Particular aspects provide novel mutant plants and plant parts thereof,
derived via mutagenesis, having disease resistance and other useful
traits. Particular embodiments provide a wheat genotype `RRR Scarlet`
(`Scarlet-Rz1`), plants and seeds thereof, methods for producing a plant
comprising crossing `Scarlet-Rz1` plants with another wheat plant, hybrid
wheat seeds and plants produced by crossing `Scarlet-Rz1` plants with
another line or plant, and creation of variants by mutagenesis or
transformation of `Scarlet-Rz1`. Additional aspects provide methods for
producing other varieties or breeding lines derived from `Scarlet-Rz1`
and to varieties or breeding lines produced thereby. Further aspects
provide for mutant plants and plant parts thereof that are resistant
and/or tolerant to plant root fungal pathogens such as Rhizoctonia and
Pythium. Additional embodiments provide mutant plants and plant parts
thereof that exhibit stress tolerance and/or resistance. Yet further
aspects provide mutant plants and plant parts thereof that are drought
resistant or tolerant.

Claims:

1. A wheat plant or a part thereof, comprising a mutation that confers
fungal tolerance derived from a root fungal pathogen-tolerant wheat
genotype resulting from chemical mutagenesis of wheat germplasm.

4. The wheat plant or part thereof of claim 1, wherein the
fungus-tolerance trait is derived by crossing a plant of the root fungal
pathogen-tolerant wheat genotype with a plant of a wheat variety that
lacks the root fungal pathogen-tolerance trait to produce progeny, and
selecting the wheat plant comprising the root fungal pathogen-tolerance
trait from the progeny.

5. The wheat plant or part thereof of claim 1, wherein the root fungal
pathogen-tolerant wheat genotype is tolerant to at least one root fungal
pathogen selected from the group consisting of Rhizoctonia and Pythium.

6. The wheat plant or part thereof of claim 5, wherein the Rhizoctonia spp
comprises at least one selected from the group consisting of R. solani
and R. oryzae.

7. The wheat plant or part thereof of claim 5, wherein the Pythium spp
comprises at least one selected from the group consisting of P. ultimum,
P. irregulars, P. debaryanum, P. aristosporum, P. volutum, and P.
sylvaticum.

9. The wheat plant or part thereof of claim 1, comprising two or more
different mutations that confer root fungal pathogen-tolerance, wherein
at least one of the two or more different mutations is derived from a
root fungal pathogen-tolerant wheat genotype resulting from chemical
mutagenesis of wheat germplasm.

10. The wheat plant or part thereof of claim 9, wherein each of the two or
more different mutations is derived from a root fungal pathogen-tolerant
wheat genotype resulting from chemical mutagenesis of wheat germplasm.

11. The wheat plant or part thereof of claim 9, wherein at least one of
the two or more different mutations comprises a semi-dominant mutation.

12. The wheat plant or part thereof of claim 9, wherein the root fungal
pathogen-tolerant wheat genotype is tolerant to at least one root fungal
pathogen selected from the group consisting of Rhizoctonia and Pythium.

14. The wheat plant or part thereof of claim 13, wherein the herbicide
consists of or comprises glyphosate or a derivative thereof.

15. The wheat plant or part thereof of claim 14, wherein the resistance to
herbicide, is derived from a glyphosate-tolerant wheat genotype selected
from the group consisting of GT-Louise, LouiseFR1-04, LouiseFR1-33,
MaconFR1-05, MaconFR1-19 and TaraFR1-07.

20. A wheat plant or part thereof produced by growing the seed of claim
19.

21. The wheat plant or part thereof of claim 20, having all the
physiological and morphological characteristics of a Scarlet-Rz1 (ATCC
Patent Deposit Number PTA-8198) genotype.

22. A method of making a root fungal pathogen-tolerant wheat genotype or
wheat plant, comprising:providing germplasm of a wheat variety;treating
the germplasm with a mutagen to produce a mutagenized germplasm;selecting
from the mutagenized germplasm a root fungal pathogen-tolerant wheat seed
comprising a genotype conferring root fungal pathogen-tolerance that is
caused by the mutagen; andgrowing a root fungal pathogen-tolerant wheat
plant from the root fungal pathogen-tolerant wheat seed.

23. The method of claim 22, wherein the germplasm consists of or comprises
a plurality of seeds.

24. The method of claim 22, wherein the mutagen is a chemical mutagen.

26. The method of claim 22, wherein the genotype conferring root fungal
pathogen-tolerance comprises at least one mutation selected from the
group consisting of a point mutation and a deletion mutation.

28. The method of claim 22, wherein the root fungal pathogen-tolerant
wheat seed is identified by growing the root fungal pathogen-tolerant
plant from the root fungal pathogen-tolerant wheat seed under conditions
suitable to expose roots thereof to a root fungal pathogen, and observing
the roots or the growth of the root fungal pathogen-tolerant plant during
or after exposure to the root fungal pathogen.

29. The method of claim 22, wherein the root fungal pathogen-tolerant
wheat genotype or plant is tolerant to at least one root fungal pathogen
selected from the group consisting of Rhizoctonia and Pythium.

30. The method of claim 29, wherein the Rhizoctonia spp comprises at least
one selected from the group consisting of R. solani and R. oryzae.

31. The method of claim 29, wherein the Pythium spp comprises at least one
selected from the group consisting of P. ultimum, P. irregulare, P.
debaryanum, P. aristosporum, P. volutum, and P. sylvaticum.

32. The method of claim 22, wherein the root fungal pathogen-tolerant
wheat plant is phenotypically similar to an unmutagenized wheat plant of
the selected wheat variety.

33. A method of making a root fungal pathogen-tolerant wheat genotype or
wheat plant, comprising:providing a plurality of seeds of a selected
wheat variety;treating the plurality of wheat seeds with a chemical
mutagen to produce a mutagenized germplasm;selecting from the plurality
of mutagenized wheat seeds a root fungal pathogen-tolerant wheat seed
comprising a genotype conferring root fungal pathogen-tolerance that is
caused by the mutagen; andgrowing a root fungal pathogen-tolerant wheat
plant from the root fungal pathogen-tolerant wheat seed, wherein the root
fungal pathogen-tolerant wheat plant is phenotypically similar to an
unmutagenized wheat plant of the selected wheat variety.

34. The method of claim 33, wherein the root fungal pathogen-tolerant
wheat genotype or plant is tolerant to at least one root fungal pathogen
selected from the group consisting of Rhizoctonia and Pythium.

35. The method of claim 34 wherein the Rhizoctonia spp comprises at least
one selected from the group consisting of R. solani and R. oryzae.

36. A method of producing a root fungal pathogen-tolerant wheat genotype
or plant, comprising:crossing a plant of a selected wheat variety with a
root fungal pathogen-tolerant wheat plant having a genotype derived from
a root fungal pathogen-tolerant wheat genotype resulting from chemical
mutagenesis of wheat germplasm, thereby producing a plurality of progeny;
andselecting a progeny that is root fungal pathogen-tolerant.

38. The method of claim 36, wherein the root fungal pathogen-tolerant
wheat genotype is that of Scarlet-Rz1 (ATCC Patent Deposit Number
PTA-8198).

39. The method of claim 36, comprising:(a) crossing plants grown from seed
of the root fungal pathogen-tolerant wheat genotype, with plants of the
selected wheat variety to produce F1 progeny plants;(b) selecting F1
progeny plants that have the root fungal pathogen-tolerance trait;(c)
crossing the selected F1 progeny plants with the plants of the selected
wheat variety to produce backcross progeny plants;(d) selecting for
backcross progeny plants that have the root fungal pathogen-tolerance
trait and physiological and morphological characteristics of said
selected wheat genotype to produce selected backcross progeny plants;
and(e) repeating steps (c) and (d) three or more times in succession to
produce selected fourth or higher backcross progeny plants that comprise
the root fungal pathogen-tolerance trait and physiological and
morphological characteristics of said selected wheat genotype as
determined at the 5% significance level when grown in the same
environmental conditions.

40. The method of claim 36, comprising:(a) crossing plants grown from seed
of the root fungal pathogen-tolerant wheat genotype, with plants of said
selected wheat variety to produce F1 progeny plants, wherein the selected
wheat variety comprises a desired trait;(b) selecting F1 progeny plants
that have the desired trait to produce selected F1 progeny plants;(c)
crossing the selected progeny plants with the plants of the root fungal
pathogen-tolerant wheat genotype to produce backcross progeny plants;(d)
selecting for backcross progeny plants that have the desired trait and
physiological and morphological characteristics of the root fungal
pathogen-tolerant wheat genotype to produce selected backcross progeny
plants; and(e) repeating steps (c) and (d) three or more times in
succession to produce selected fourth or higher backcross progeny plants
that comprise the desired trait and physiological and morphological
characteristics of said root fungal pathogen-tolerant wheat genotype as
determined at the 5% significance level when grown in the same
environmental conditions.

42. The method of claim 40, wherein the herbicide consists of or comprises
glyphosate or a derivative thereof.

43. The method of claim 42, wherein the resistance to herbicide, is
derived from a glyphosate-tolerant wheat genotype selected from the group
consisting of GT-Louise, LouiseFR1-04, LouiseFR1-33, MaconFR1-05,
MaconFR1-19 and TaraFR1-07.

44. A method of producing a root fungal pathogen-tolerant wheat genotype
or plant, comprising;providing a selected wheat variety; andintroducing
into the selected wheat variety using suitable methods a transgene
comprising a mutation that confers fungal tolerance, the mutation derived
from a root fungal pathogen-tolerant wheat genotype resulting from
chemical mutagenesis of wheat germplasm.

46. The method of claim 44, wherein the root fungal pathogen-tolerant
wheat genotype from which the mutation is derived is Scarlet-Rz1 (ATCC
Patent Deposit Number PTA-8198).

47. A transgenic plant obtained using the method of claim 44.

48. A method of making a drought-tolerant wheat genotype or wheat plant,
comprising:providing germplasm of a wheat variety;treating the germplasm
with a mutagen to produce a mutagenized germplasm;selecting from the
mutagenized germplasm a drought-tolerant wheat seed comprising a genotype
conferring drought-tolerance that is caused by the mutagen; andgrowing a
drought-tolerant wheat plant from the drought-tolerant wheat seed.

49. The method of claim 48, wherein the germplasm consists of or comprises
a plurality of seeds.

50. The method of claim 48, wherein the mutagen is a chemical mutagen.

52. The method of claim 48, wherein the drought-tolerant wheat seed is
identified based on increased sensitivity to the plant hormone ABA
(abscisic acid) during seed germination.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application claims the benefit of priority from U.S.
Provisional Patent Application Ser. No. 60/771,402, filed 7 Feb. 2006 and
entitled "MUTATION BREEDING FOR RESISTANCE TO DISEASE AND OTHER USEFUL
TRAITS," and 60/771,285, filed 7 Feb. 2006 and entitled
"GLYPHOSATE-TOLERANT WHEAT GENOTYPE," both of which are incorporated
herein by reference.

[0003]2,500 wheat seeds derived from selection Scarlet-Rz1 (described
herein) were named `Scarlet RZ-1` (or `Scarlet-Rz1`), were increased by
self-pollination, and were deposited on 6 Feb. 2007 at 9:39 AM under the
terms of the Budapest Treaty at the American Type Culture Collection,
10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A., and have
received ATCC Accession No. PTA-______. Seeds from this deposit will be
irrevocably made available upon the grant of a patent that makes
reference to this deposit. However, the availability of these seeds is
not to be construed as a license to practice the claimed invention in
contravention of rights granted under the authority of any government in
accordance with its patent or breeder's right laws.

BACKGROUND

[0004]Wheat is grown worldwide and is the most widely adapted cereal.
There are five main wheat market classes. They include the four common
wheat (Triticum aestivum L.) classes: hard red winter, hard red spring,
soft red winter, and white. The fifth class is durum (Triticum turgidum
L.). Common wheats are used in a variety of food products such as bread,
cookies, cakes, crackers, and noodles. In general the hard wheat classes
are milled into flour used for breads and the soft wheat classes are
milled into flour used for pastries and crackers. Wheat starch is used in
the food and paper industries, as laundry starches, and in other
products. Because of its use in baking, the grain quality of wheat is
very important. To test the grain quality of wheat for use as flour,
milling properties are analyzed. Important milling properties are
relative hardness or softness, weight per bushel of wheat (test weight),
siftability of the flour, break flour yield, middlings flour yield, total
flour yield, flour ash content, and wheat-to-flour protein conversion.
Good processing quality for flour is also important. Good quality
characteristics for flour from soft wheats include low to medium-low
protein content, low water absorption, production of large-diameter test
cookies and large volume cakes. Wheat glutenins and gliadins, which
together confer the properties of elasticity and extensibility, play an
important role in the grain quality. Changes in quality and quantity of
these proteins change the end product for which the wheat can be used.

[0006]Mutation Breeding. Mutation Breeding comprises the use of chemical
mutagenesis to increase genetic diversity. Natural mutations arise due to
errors in replicating DNA. Such mutations are exploited when they are
introduced from wild relatives of crop plants. The error rate during DNA
replication is increased by treatment of plant seeds with chemicals
called mutagens, and this chemical mutagenesis is a tool for increasing
the variation in a plant population. Chemical-induced variants are
currently accepted as an alternative to "Genetically Modified" plants
made by transformation. Over 2,250 crop varieties now in use come from
mutation breeding (M. J. Chrispeels and D. E. Sadava 2003, Plants, Genes,
and Crop Biotechnology (2nd edition), Jones and Bartlett Publishers
(Boston). Currently, for example, the herbicide-resistant Clearfield
Wheat is a well-known example of a wheat variety from mutation breeding.

[0007]There is a pronounced need in the art for root rot resistant plants
(e.g., wheat). There is a pronounced need in the art Rhizoctonia root rot
resistant plants (e.g., wheat). There is a pronounced need in the art for
novel methods for generating such resistant plants. There is a pronounced
need in the art for novel methods comprising mutation breeding used to
address the major problems that occur in wheat production (e.g.,
Rhizoctonia root rot).

SUMMARY OF PARTICULAR ASPECTS OF THE INVENTION

[0008]Particular aspect relate to a novel and distinctive wheat variety
genotype, designated `RRR Scarlet` or `Rz1,` which is result careful
breeding and selection in a wheat mutation breeding program.

[0011]Further embodiments provide methods for producing a wheat plant
produced by crossing the variety genotype `RRR Scarlet` (`Rz1`) with
another wheat plant, and hybrid wheat seeds and plants produced by
crossing the genotype `RRR Scarlet` (`Rz1`) with another wheat line or
plant, and the creation of variants by mutagenesis or transformation of
genotype `RRR Scarlet` (`Rz1`).

[0012]Additional aspects comprise methods for producing other wheat
varieties or breeding lines derived from wheat variety `RRR Scarlet`
(`Rz1`) and to wheat varieties or breeding lines produced by those
methods.

[0013]Further aspects provide for mutant plants and plant parts thereof
that are resistant and/or tolerant to plant pathogens such as Rhizoctonia
and Pythium. As used herein, the term "plant parts" includes plant
protoplasts, plant cell tissue cultures from which wheat plants can be
regenerated, plant calli, plant clumps, and plant cells that are intact
in plants or parts of plants, such as embryos, pollen, ovules, pericarp,
seed, flowers, florets, heads, spikes, leaves, roots, root tips, anthers,
and the like. The term also includes products of a plant, including but
not limited to flour, starch, oil, wheat germ, and so on.

[0016]Additional embodiments comprise methods for the recovery of a novel
genetic change in gene(s) conferring the desired response (resistance
gene) `RRR Scarlet` (`Rz1`) with little disruption to the remaining
genetic pathways. Thus, according to particular aspects, mutated
cultivars (e.g., Zak and Scarlet) and plants with resistance are
genetically similar to the respective parent with the advantage of the
new resistance gene.

[0017]Particular preferred aspects provide a wheat plant or a part
thereof, comprising a mutation that confers fungal tolerance derived from
a root fungal pathogen-tolerant wheat genotype resulting from chemical
mutagenesis of wheat germplasm. In certain embodiments, the chemical
mutagenesis comprises treatment of wheat seeds with ethyl methane
sulfonate (EMS). In particular embodiments, the root fungal
pathogen-tolerant wheat genotype is Scarlet-Rz1 (ATCC Accession No.). In
particular aspects, the fungus-tolerance trait is derived by crossing a
plant of the root fungal pathogen-tolerant wheat genotype with a plant of
a wheat variety that lacks the root fungal pathogen-tolerance trait to
produce progeny, and selecting the wheat plant comprising the root fungal
pathogen-tolerance trait from the progeny.

[0018]In certain aspects, the root fungal pathogen-tolerant wheat genotype
is tolerant to at least one root fungal pathogen selected from the group
consisting of Rhizoctonia and Pythium. In particular embodiments, the
Rhizoctonia spp comprises at least one selected from the group consisting
of R. solani and R. oryzae. In certain embodiments, the Pythium spp
comprises at least one selected from the group consisting of P. ultimum,
P. irregulare, P. debaryanum, P. aristosporum, P. volutum, and P.
sylvaticum.

[0019]In particular embodiments, the root fungal pathogen-tolerant wheat
genotype comprises a semi-dominant mutation. In certain aspects, the
wheat plant or part thereof comprises two or more different mutations
that confer root fungal pathogen-tolerance, wherein at least one of the
two or more different mutations is derived from a root fungal
pathogen-tolerant wheat genotype resulting from chemical mutagenesis of
wheat germplasm. In particular aspects, each of the two or more different
mutations is derived from a root fungal pathogen-tolerant wheat genotype
resulting from chemical mutagenesis of wheat germplasm. In particular
aspects, at least one of the two or more different mutations comprises a
semi-dominant mutation. In particular embodiments, the root fungal
pathogen-tolerant wheat genotype is tolerant to at least one root fungal
pathogen selected from the group consisting of Rhizoctonia and Pythium.

[0020]In particular aspects, the wheat plant or part thereof further
comprises at least one trait selected from the group consisting of: male
sterility, resistance to an herbicide, insect resistance, disease
resistance; waxy starch; modified fatty acid metabolism, modified phytic
acid metabolism, modified carbohydrate metabolism, modified waxy starch
content, modified gluten content, and modified water stress tolerance. In
certain aspects, the herbicide consists of or comprises glyphosate or a
derivative thereof. In particular embodiments, the resistance to
herbicide, is derived from a glyphosate-tolerant wheat genotype selected
from the group consisting of GT-Louise, LouiseFR1-04, LouiseFR1-33,
MaconFR1-05, MaconFR1-19 and TaraFR1-07.

[0022]Additional aspects provide a true-breeding seed of any of the above
described plants. Further embodiments provide a wheat plant or part
thereof produced by growing the seed of claim 19. Yet additional aspects
provide a wheat plant or part thereof having all the physiological and
morphological characteristics of a Scarlet-Rz1 (ATCC Accession No.)
genotype.

[0023]Further aspects provide a method of making a root fungal
pathogen-tolerant wheat genotype or wheat plant, comprising: providing
germplasm of a wheat variety; treating the germplasm with a mutagen to
produce a mutagenized germplasm; selecting from the mutagenized germplasm
a root fungal pathogen-tolerant wheat seed comprising a genotype
conferring root fungal pathogen-tolerance that is caused by the mutagen;
and growing a root fungal pathogen-tolerant wheat plant from the root
fungal pathogen-tolerant wheat seed. In certain aspects the germplasm
comprises a plurality of seeds. In additional aspect the germplasm
comprises wheat microspores. In certain embodiments, the mutagen is a
chemical mutagen. In particular embodiments, the chemical mutagen is
ethyl methane sulfonate (EMS). In particular aspects, the genotype
conferring root fungal pathogen-tolerance comprises at least one mutation
selected from the group consisting of a point mutation and a deletion
mutation. In certain embodiments, the genotype conferring root fungal
pathogen-tolerance comprises a semi-dominant mutation. In particular
aspects, the root fungal pathogen-tolerant wheat seed is identified by
growing the root fungal pathogen-tolerant plant from the root fungal
pathogen-tolerant wheat seed under conditions suitable to expose roots
thereof to a root fungal pathogen, and observing the roots or the growth
of the root fungal pathogen-tolerant plant during or after exposure to
the root fungal pathogen. In certain embodiments, the root fungal
pathogen-tolerant wheat genotype or plant is tolerant to at least one
root fungal pathogen selected from the group consisting of Rhizoctonia
and Pythium. In certain embodiments, the Rhizoctonia spp comprises at
least one selected from the group consisting of R. solani and R. oryzae.
In particular embodiments, the Pythium spp comprises at least one
selected from the group consisting of P. ultimum, P. irregulare, P.
debaryanum, P. aristosporum, P. volutum, and P. sylvaticum. In certain
embodiments, the root fungal pathogen-tolerant wheat plant is
phenotypically similar to an unmutagenized wheat plant of the selected
wheat variety.

[0024]Yet further aspects, provide a method of making a root fungal
pathogen-tolerant wheat genotype or wheat plant, comprising: providing a
plurality of seeds of a selected wheat variety; treating the plurality of
wheat seeds with a chemical mutagen to produce a mutagenized germplasm;
selecting from the plurality of mutagenized wheat seeds a root fungal
pathogen-tolerant wheat seed comprising a genotype conferring root fungal
pathogen-tolerance that is caused by the mutagen; and growing a root
fungal pathogen-tolerant wheat plant from the root fungal
pathogen-tolerant wheat seed, wherein the root fungal pathogen-tolerant
wheat plant is phenotypically similar to an unmutagenized wheat plant of
the selected wheat variety. In certain embodiments, the root fungal
pathogen-tolerant wheat genotype or plant is tolerant to at least one
root fungal pathogen selected from the group consisting of Rhizoctonia
and Pythium. In particular embodiments, the Rhizoctonia spp comprises at
least one selected from the group consisting of R. solani and R. oryzae.

[0025]Additional embodiments provide a method of producing a root fungal
pathogen-tolerant wheat genotype or plant, comprising: crossing a plant
of a selected wheat variety with a root fungal pathogen-tolerant wheat
plant having a genotype derived from a root fungal pathogen-tolerant
wheat genotype resulting from chemical mutagenesis of wheat germplasm,
thereby producing a plurality of progeny; and selecting a progeny that is
root fungal pathogen-tolerant. In certain embodiments, the chemical
mutagenesis comprises treatment of wheat seeds with ethyl methane
sulfonate (EMS). In certain embodiments, the root fungal
pathogen-tolerant wheat genotype is that of Scarlet-Rz1 (ATCC Accession
No.).

[0026]In certain embodiments, the method comprises: (a) crossing plants
grown from seed of the root fungal pathogen-tolerant wheat genotype, with
plants of the selected wheat variety to produce F1 progeny plants; (b)
selecting F1 progeny plants that have the root fungal pathogen-tolerance
trait; (c) crossing the selected F1 progeny plants with the plants of the
selected wheat variety to produce backcross progeny plants; (d) selecting
for backcross progeny plants that have the root fungal pathogen-tolerance
trait and physiological and morphological characteristics of said
selected wheat genotype to produce selected backcross progeny plants; and
(e) repeating steps (c) and (d) three or more times in succession to
produce selected fourth or higher backcross progeny plants that comprise
the root fungal pathogen-tolerance trait and physiological and
morphological characteristics of said selected wheat genotype as
determined at the 5% significance level when grown in the same
environmental conditions.

[0028]In certain embodiments of the methods, the herbicide consists of or
comprises glyphosate or a derivative thereof. In particular aspects, the
resistance to herbicide, is derived from a glyphosate-tolerant wheat
genotype selected from the group consisting of GT-Louise, LouiseFR1-04,
LouiseFR1-33, MaconFR1-05, MaconFR1-19 and TaraFR1-07.

[0030]Additional aspects proved a method of making a drought-tolerant
wheat genotype or wheat plant, comprising: providing germplasm of a wheat
variety; treating the germplasm with a mutagen to produce a mutagenized
germplasm; selecting from the mutagenized germplasm a drought-tolerant
wheat seed comprising a genotype conferring drought-tolerance that is
caused by the mutagen; and growing a drought-tolerant wheat plant from
the drought-tolerant wheat seed. In certain embodiments, the germplasm
comprises a plurality of seeds. In particular embodiments, the mutagen is
a chemical mutagen. In certain aspects, the chemical mutagen comprises or
consists of ethyl methane sulfonate (EMS). In particular embodiments, the
drought-tolerant wheat seed is identified based on increased sensitivity
to the plant hormone ABA (abscisic acid) during seed germination.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 shows, according to particular exemplary embodiments of the
present invention, growth chamber evaluated wheat seedlings with and
without Rhizoctonia solani inoculum at the M1 stage. The resistant mutant
RRR Scarlet is in the two containers in the far left of the photograph, a
susceptible mutant is found in containers 3 and 4, and the control
Scarlet, with and without Rhizoctonia inoculum, is found in containers 5
and 6, and 7 and 8, respectively. Seedling height is significantly
correlated with disease infection levels. RRR Scarlet is similar in
height to the non-inoculated control. RRR Scarlet is variously referred
to as Resistant Mutant, Scarlet Mutant Line 15, S 015 or S 015-6-4 in the
figure.

[0032]FIG. 2 shows, according to particular exemplary embodiments of the
present invention, growth chamber evaluated wheat seedling with and
without Rhizoctonia solani inoculum at the BC1F3
(pedigree=Scarlet2*RRR Scarlet) stage. Reading from left to right,
BC1 derivatives of RRR Scarlet are found in cones 1 and 2,
inoculated Scarlet is in cones 3 and 4, BC1 derivatives of a
susceptible mutant are found in cones 5 and 6, and un-inoculated Scarlet
is found in cones 7 and 8. RRR Scarlet is similar in height to the
un-inoculated control indicating that this genotype is tolerant to the
pathogen, and that this resistance is heritable. RRR Scarlet is variously
referred to as Resistant Mutant, Scarlet Mutant Line 15, S 015 or S
015-6-4 in the figure.

[0033]FIG. 3 shows, according to particular exemplary embodiments of the
present invention, root scans, which reflect root health, of Scarlet and
RRR Scarlet treated with or without Rhizoctonia solani. Large root mass
is associated with low disease levels. A) Scarlet without inoculum; B)
Scarlet with inoculum; C) Scarlet-015-6-4 with inoculum; and D) highly
susceptible mutant line with inoculum. The root mass of inoculated
Scarlet-015-6-4 is similar to that of un-inoculated Scarlet. RRR Scarlet
is variously referred to as Resistant Mutant, Scarlet Mutant Line 15, S
015 or S 015-6-4 in the figure.

[0034]FIG. 4 shows, according to particular exemplary embodiments of the
present invention, frequency of seedling tolerance in three generations
of Scarlet Rz1, indicated by disease severity scores.

[0035]FIG. 5 shows, according to particular exemplary embodiments of the
present invention, average root length values obtained from individuals
of the first (BC1F2) and second (BC2F3) backcross groups of Scarlet Rz1,
wild type Scarlet (Wt) and non-inoculated wild type Scarlet control
("cont").

[0036]FIG. 6 shows, according to particular exemplary embodiments of the
present invention, comparison of distribution of seedling weights of
individuals from three BC2F3 groups of Scarlet Rz1.

[0038]FIG. 8 shows, according to particular exemplary embodiments of the
present invention, comparison of shoot height in 14-day-old Scarlet wild
type (Wt), plants of BC2F3 group 20-6R14, and susceptible sibs of
20-6R14, all used for FIG. 7.

[0039]FIGS. 9A and B show, according to particular exemplary embodiments
of the present invention, seedling tolerance of BC2F4 group 20-6R14 to R.
solani AG-8 isolate C1

[0040]FIGS. 10A and B show, according to particular exemplary embodiments
of the present invention, seedling tolerance of BC2F4 group 20-6R14 to R.
oryzae isolate 0801387

[0041]FIGS. 11A, B and C show, according to particular exemplary
embodiments of the present invention, damping-off tolerance to R. oryzae
in BC2F4 individuals of group 20-6R14 of Scarlet Rz1

[0042]FIG. 12 shows, according to particular exemplary embodiments of the
present invention, Pythium tolerance in Scarlet Rz1 as indicated by
distribution of first leaf length (rum) among individuals of Scarlet wild
type (Wt) and Scarlet Rz1 BC1F2 populations P20-6 and P21-3. Seedlings
were germinated for 2 to 4 days, then grown for 14 days in pasteurized
Spillman soil infested with 1000 propagules per gram (ppg) each of
Pythium ultimum isolate 0900119 and 1000 ppg P. irregulare grp I isolate
0900101.

[0043]FIG. 13 shows, according to particular exemplary embodiments of the
present invention, that a proportion of plants within a BC1F2 group are
expected to be heterozygous for Pythium tolerance and show an
intermediate degree of tolerance, whereas some plants will be homozygous
for either tolerance (strong tolerance) or susceptibility (no tolerance).

[0044]FIG. 14 shows, according to particular exemplary embodiments of the
present invention, to validate the Pythium tolerance indicated by leaf
measurements, plants from the R (strong tolerance) and S (susceptible)
leaf length classes (see FIG. 13) were picked at random for root length
analysis. The findings show that Pythium tolerance in BC1F2 plants of
Scarlet Rz1 is indicated by enhanced root growth, as well as enhanced
foliar growth.

[0046]FIG. 16 shows, according to particular exemplary embodiments of the
present invention, seed increase of Rz1 in the Wheat Research Facility at
Washington State University in Pullman. Phenotypically, Rz1 is
indistinguishable from non-mutagenized spring wheat under typical
greenhouse growth conditions. Plants are vigorous, healthy and have high
fertility levels.

DETAILED DESCRIPTION OF THE INVENTION

[0047]Embodiments of the invention comprise the treatment of a wheat plant
with a mutagen and the plant produced by mutagenesis of the wheat plant.
Information about mutagens and mutagenizing seeds or pollen are presented
in the IAEA's Manual on Mutation Breeding (IAEA, 1977) other information
about mutation breeding in wheat can be found in C. F. Konzak, "Mutations
and Mutation Breeding" chapter 7B, of Wheat and Wheat Improvement,
2nd edition, ed. Heyne, 1987.

Exemplary Preferred Embodiments

[0048]Particular aspects provide a wheat plant or a part thereof,
comprising a mutation that confers fungal tolerance derived from a root
fungal pathogen-tolerant wheat genotype resulting from chemical
mutagenesis of wheat germplasm. In certain embodiments, the chemical
mutagenesis comprises treatment of wheat seeds with ethyl methane
sulfonate (EMS). In particular embodiments, the root fungal
pathogen-tolerant wheat genotype is Scarlet-Rz1 (ATCC Accession No.). In
particular aspects, the fungus-tolerance trait is derived by crossing a
plant of the root fungal pathogen-tolerant wheat genotype with a plant of
a wheat variety that lacks the root fungal pathogen-tolerance trait to
produce progeny, and selecting the wheat plant comprising the root fungal
pathogen-tolerance trait from the progeny.

[0049]In certain aspects, the root fungal pathogen-tolerant wheat genotype
is tolerant to at least one root fungal pathogen selected from the group
consisting of Rhizoctonia and Pythium. In particular embodiments, the
Rhizoctonia spp comprises at least one selected from the group consisting
of R. solani and R. oryzae. In certain embodiments, the Pythium spp
comprises at least one selected from the group consisting of P. ultimum,
P. irregulare, P. debaryanum, P. aristosporum, P. volutum, and P.
sylvaticum.

[0050]In particular embodiments, the root fungal pathogen-tolerant wheat
genotype comprises a semi-dominant mutation. In certain aspects, the
wheat plant or part thereof comprises two or more different mutations
that confer root fungal pathogen-tolerance, wherein at least one of the
two or more different mutations is derived from a root fungal
pathogen-tolerant wheat genotype resulting from chemical mutagenesis of
wheat germplasm. In particular aspects, each of the two or more different
mutations is derived from a root fungal pathogen-tolerant wheat genotype
resulting from chemical mutagenesis of wheat germplasm. In particular
aspects, at least one of the two or more different mutations comprises a
semi-dominant mutation. In particular embodiments, the root fungal
pathogen-tolerant wheat genotype is tolerant to at least one root fungal
pathogen selected from the group consisting of Rhizoctonia and Pythium.

[0051]In particular aspects, the wheat plant or part thereof further
comprises at least one trait selected from the group consisting of: male
sterility, resistance to an herbicide, insect resistance, disease
resistance; waxy starch; modified fatty acid metabolism, modified phytic
acid metabolism, modified carbohydrate metabolism, modified waxy starch
content, modified gluten content, and modified water stress tolerance. In
certain aspects, the herbicide consists of or comprises glyphosate or a
derivative thereof. In particular embodiments, the resistance to
herbicide, is derived from a glyphosate-tolerant wheat genotype selected
from the group consisting of GT-Louise, LouiseFR1-04, LouiseFR1-33,
MaconFR1-05, MaconFR1-19 and TaraFR1-07.

[0053]Additional aspects provide a true-breeding seed of any of the above
described plants.

[0054]Further embodiments provide a wheat plant or part thereof produced
by growing the seed of claim 19.

[0055]Yet additional aspects provide a wheat plant or part thereof having
all the physiological and morphological characteristics of a Scarlet-Rz1
(ATCC Accession No.) genotype.

[0056]Further aspects provide a method of making a root fungal
pathogen-tolerant wheat genotype or wheat plant, comprising: providing
germplasm of a wheat variety; treating the germplasm with a mutagen to
produce a mutagenized germplasm; selecting from the mutagenized germplasm
a root fungal pathogen-tolerant wheat seed comprising a genotype
conferring root fungal pathogen-tolerance that is caused by the mutagen;
and growing a root fungal pathogen-tolerant wheat plant from the root
fungal pathogen-tolerant wheat seed. In certain aspects the germplasm
comprises a plurality of seeds. In additional aspect the germplasm
comprises wheat microspores. In certain embodiments, the mutagen is a
chemical mutagen. In particular embodiments, the chemical mutagen is
ethyl methane sulfonate (EMS). In particular aspects, the genotype
conferring root fungal pathogen-tolerance comprises at least one mutation
selected from the group consisting of a point mutation and a deletion
mutation. In certain embodiments, the genotype conferring root fungal
pathogen-tolerance comprises a semi-dominant mutation. In particular
aspects, the root fungal pathogen-tolerant wheat seed is identified by
growing the root fungal pathogen-tolerant plant from the root fungal
pathogen-tolerant wheat seed under conditions suitable to expose roots
thereof to a root fungal pathogen, and observing the roots or the growth
of the root fungal pathogen-tolerant plant during or after exposure to
the root fungal pathogen. In certain embodiments, the root fungal
pathogen-tolerant wheat genotype or plant is tolerant to at least one
root fungal pathogen selected from the group consisting of Rhizoctonia
and Pythium. In certain embodiments, the Rhizoctonia spp comprises at
least one selected from the group consisting of R. solani and R. oryzae.
In particular embodiments, the Pythium spp comprises at least one
selected from the group consisting of P. ultimum, P. irregulare, P.
debaryanum, P. aristosporum, P. volutum, and P. sylvaticum. In certain
embodiments, the root fungal pathogen-tolerant wheat plant is
phenotypically similar to an unmutagenized wheat plant of the selected
wheat variety.

[0057]Yet further aspects, provide a method of making a root fungal
pathogen-tolerant wheat genotype or wheat plant, comprising: providing a
plurality of seeds of a selected wheat variety; treating the plurality of
wheat seeds with a chemical mutagen to produce a mutagenized germplasm;
selecting from the plurality of mutagenized wheat seeds a root fungal
pathogen-tolerant wheat seed comprising a genotype conferring root fungal
pathogen-tolerance that is caused by the mutagen; and growing a root
fungal pathogen-tolerant wheat plant from the root fungal
pathogen-tolerant wheat seed, wherein the root fungal pathogen-tolerant
wheat plant is phenotypically similar to an unmutagenized wheat plant of
the selected wheat variety. In certain embodiments, the root fungal
pathogen-tolerant wheat genotype or plant is tolerant to at least one
root fungal pathogen selected from the group consisting of Rhizoctonia
and Pythium. In particular embodiments, the Rhizoctonia spp comprises at
least one selected from the group consisting of R. solani and R. oryzae.

[0058]Additional embodiments provide a method of producing a root fungal
pathogen-tolerant wheat genotype or plant, comprising: crossing a plant
of a selected wheat variety with a root fungal pathogen-tolerant wheat
plant having a genotype derived from a root fungal pathogen-tolerant
wheat genotype resulting from chemical mutagenesis of wheat germplasm,
thereby producing a plurality of progeny; and selecting a progeny that is
root fungal pathogen-tolerant. In certain embodiments, the chemical
mutagenesis comprises treatment of wheat seeds with ethyl methane
sulfonate (EMS). In certain embodiments, the root fungal
pathogen-tolerant wheat genotype is that of Scarlet-Rz1 (ATCC Accession
No.).

[0059]In certain embodiments, the method comprises: (a) crossing plants
grown from seed of the root fungal pathogen-tolerant wheat genotype, with
plants of the selected wheat variety to produce F1 progeny plants; (b)
selecting F1 progeny plants that have the root fungal pathogen-tolerance
trait; (c) crossing the selected F1 progeny plants with the plants of the
selected wheat variety to produce backcross progeny plants; (d) selecting
for backcross progeny plants that have the root fungal pathogen-tolerance
trait and physiological and morphological characteristics of said
selected wheat genotype to produce selected backcross progeny plants; and
(e) repeating steps (c) and (d) three or more times in succession to
produce selected fourth or higher backcross progeny plants that comprise
the root fungal pathogen-tolerance trait and physiological and
morphological characteristics of said selected wheat genotype as
determined at the 5% significance level when grown in the same
environmental conditions.

[0061]In certain embodiments of the methods, the herbicide consists of or
comprises glyphosate or a derivative thereof. In particular aspects, the
resistance to herbicide, is derived from a glyphosate-tolerant wheat
genotype selected from the group consisting of GT-Louise, LouiseFR1-04,
LouiseFR1-33, MaconFR1-05, MaconFR1-19 and TaraFR1-07.

[0063]Additional aspects proved a method of making a drought-tolerant
wheat genotype or wheat plant, comprising: providing germplasm of a wheat
variety; treating the germplasm with a mutagen to produce a mutagenized
germplasm; selecting from the mutagenized germplasm a drought-tolerant
wheat seed comprising a genotype conferring drought-tolerance that is
caused by the mutagen; and growing a drought-tolerant wheat plant from
the drought-tolerant wheat seed. In certain embodiments, the germplasm
comprises a plurality of seeds. In particular embodiments, the mutagen is
a chemical mutagen. In certain aspects, the chemical mutagen comprises or
consists of ethyl methane sulfonate (EMS). In particular embodiments, the
drought-tolerant wheat seed is identified based on increased sensitivity
to the plant hormone ABA (abscisic acid) during seed germination.

[0065]A further embodiment comprises or is a method of developing a
backcross conversion `RRR Scarlet` (`Rz1`) plant that involves the
repeated backcrossing to wheat variety `RRR Scarlet` (`Rz1`). The number
of backcrosses made may be 2, 3, 4, 5, 6 or greater, and the specific
number of backcrosses used will depend upon the genetics of the donor
parent and whether molecular markers are utilized in the backcrossing
program. See, for example, R. E. Allan, "Wheat" in Principles of Cultivar
Development, Fehr, W. R. Ed. (Macmillan Publishing Company, New York,
1987) pages 722-723, incorporated herein by reference. Using backcrossing
methods, one of ordinary skill in the art can develop individual plants
and populations of plants that retain at least 70%, 75%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% of the genetic profile of variety RRR Scarlet. The
percentage of the genetics retained in the backcross conversion may be
measured by either pedigree analysis or through the use of genetic
techniques such as molecular markers or electrophoresis. In pedigree
analysis, on average 50% of the starting germplasm would be passed to the
progeny line after one cross to another line, 75% after backcrossing
once, 87.5% after backcrossing twice, and so on. Molecular markers could
also be used to confirm and/or determine the recurrent parent used. The
backcross conversion developed from this method may be similar to variety
`RRR Scarlet` (`Rz1`) for the results listed in TABLE 1. Such similarity
may be measured by a side by side phenotypic comparison, with differences
and similarities determined at a 5% significance level. Any such
comparison should be made in environmental conditions that account for
the trait being transferred. For example, herbicide should not be applied
in the phenotypic comparison of herbicide resistant backcross conversion
of `RRR Scarlet` (`Rz1`) to variety `RRR Scarlet` (`Rz1`).

Essentially Derived Varieties

[0066]Another embodiment of the invention is an essentially derived
variety of genotype `RRR Scarlet` (`Rz1`). As determined by the UPOV
Convention, essentially derived varieties may be obtained for example by
the selection of a natural or induced mutant, or of a somaclonal variant,
the selection of a variant individual from plants of the initial variety,
backcrossing, or transformation by genetic engineering. An essentially
derived variety of genotype `RRR Scarlet` (`Rz1`) is further defined as
one whose production requires the repeated use of genotype `RRR Scarlet`
(`Rz1`) or is predominately derived from genotype of variety `RRR
Scarlet` (`Rz1`). International Convention for the Protection of New
Varieties of Plants, as amended on Mar. 19, 1991, Chapter V, Article 14,
Section 5(c).

Plant Breeding

[0067]Additional aspects comprise methods for using wheat variety `RRR
Scarlet` (`Rz1`) in plant breeding. One such embodiment is the method of
crossing wheat variety `RRR Scarlet` (`Rz1`) with another variety of
wheat to form a first generation population of F1 plants. The population
of first generation F1 plants produced by this method is also an
embodiment of the invention. This first generation population of F1
plants will comprise an essentially complete set of the alleles of wheat
variety `RRR Scarlet` (`Rz1`). One of ordinary skill in the art can
utilize either breeder books or molecular methods to identify a
particular F1 plant produced using wheat variety `RRR Scarlet` (`Rz1`),
and any such individual plant is also encompassed by this invention.
These embodiments also cover use of transgenic or backcross conversions
of wheat variety `RRR Scarlet` (`Rz1`) to produce first generation F1
plants.

[0068]Yet additional aspects comprise a method of developing a `RRR
Scarlet` (`Rz1`)-progeny wheat plant comprising crossing variety `RRR
Scarlet` (`Rz1`) with a second wheat plant and performing a breeding
method is also an embodiment of the invention. A specific method for
producing a line derived from wheat variety `RRR Scarlet` (`Rz1`) is as
follows. One of ordinary skill in the art would cross wheat variety `RRR
Scarlet` (`Rz1`) with another variety of wheat, such as an elite variety.
The F1 seed derived from this cross would be grown to form a homogeneous
population. The F1 seed would contain one set of the alleles from variety
`RRR Scarlet` (`Rz1`) and one set of the alleles from the other wheat
variety. The F1 genome would be made-up of 50% variety RRR Scarlet and
50% of the other elite variety. The F1 seed would be grown and allowed to
self, thereby forming F2 seed. On average the F2 seed would have derived
50% of its alleles from the genotype of `RRR Scarlet` (`Rz1`) and 50%
from the other wheat variety, but various individual plants from the
population would have a much greater percentage of their alleles derived
variety `RRR Scarlet` (`Rz1`) (Wang J. and R. Bernardo, 2000, Crop Sci.
40:659-665 and Bernardo, R. and A. L. Kahler, 2001, Theor. Appl. Genet
102:986-992). The F2 seed would be grown and selection of plants would be
made based on visual observation and/or measurement of traits. The `RRR
Scarlet` (`Rz1`)-derived progeny that exhibit one or more of the desired
`RRR Scarlet` (`Rz1`)-derived traits would be selected and each plant
would be harvested separately. This F3 seed from each plant would be
grown in individual rows and allowed to self. Then selected rows or
plants from the rows would be harvested and threshed individually. The
selections would again be based on visual observation and/or measurements
for desirable traits of the plants, such as one or more of the desirable
`RRR Scarlet` (`Rz1`)-derived traits. The process of growing and
selection would be repeated any number of times until a homozygous `RRR
Scarlet` (`Rz1`)-derived wheat plant is obtained. The homozygous `RRR
Scarlet` (`Rz1`)-derived wheat plant would contain desirable traits
derived from wheat genotype `RRR Scarlet` (`Rz1`), some of which may not
have been expressed by the other original wheat variety to which wheat
genotype `RRR Scarlet` (`Rz1`) was crossed and some of which may have
been expressed by both wheat varieties but now would be at a level equal
to or greater than the level expressed in wheat genotype RRR Scarlet. The
homozygous `RRR Scarlet` (`Rz1`)-derived wheat plants would have, on
average, 50% of their genes derived from wheat variety genotype `RRR
Scarlet` (`Rz1`), but various individual plants from the population would
have a much greater percentage of their alleles derived from genotype of
`RRR Scarlet` (`Rz1`). The breeding process, of crossing, selfing, and
selection may be repeated to produce another population of `RRR Scarlet`
(`Rz1`)-derived wheat plants with, on average, 25% of their genes derived
from wheat `RRR Scarlet` (`Rz1`), but various individual plants from the
population would have a much greater percentage of their alleles derived
from genotype `RRR Scarlet` (`Rz1`). Another embodiment comprises or is a
homozygous RRR Scarlet-derived wheat plant that has received RRR
Scarlet-derived traits.

[0069]The previous example can be modified in numerous ways, for instance
selection may or may not occur at every selfing generation, selection may
occur before or after the actual self-pollination process occurs, or
individual selections may be made by harvesting individual spikes,
plants, rows or plots at any point during the breeding process described.
In addition, double haploid breeding methods may be used at any step in
the process. The population of plants produced at each and any generation
of selfing is also an embodiment of the invention, and each such
population variety `RRR Scarlet` (`Rz1`), 25% of its genes from wheat
variety `RRR Scarlet` (`Rz1`) in the second cycle of crossing, selfing,
and selection, 12.5% of its genes from wheat variety `RRR Scarlet`
(`Rz1`) in the third cycle of crossing, selfing, and selection, and so
on.

[0070]Another embodiment of this invention is the method of crossing
plants of the genotype `RRR Scarlet` (`Rz1`) with another variety of
wheat and applying double haploid methods to the F1 seed or F1 plant or
to any generation of `RRR Scarlet` (`Rz1`) -derived wheat obtained by the
selfing of this cross.

[0071]Further aspects are directed to methods for producing `RRR Scarlet`
(`Rz1`)-derived wheat plants by crossing wheat variety `RRR Scarlet`
(`Rz1`) with a wheat plant and growing the progeny seed, and repeating
the crossing or selfing along with the growing steps with the `RRR
Scarlet` (`Rz1`)-derived wheat plant from 1 to 2 times, 1 to 3 times, 1
to 4 times, or 1 to 5 times. Thus, any and all methods using wheat
variety `RRR Scarlet` (`Rz1`) in breeding are part of this invention,
including selfing, pedigree breeding, backcrossing, hybrid production and
crosses to populations. Unique starch profiles, molecular marker profiles
and/or breeding records can be used by those of ordinary skill in the art
to identify the progeny lines or populations derived from these breeding
methods.

[0072]In addition, this invention also encompasses progeny with the same
or greater yield of `RRR Scarlet` (`Rz1`), the same or greater drought
tolerance of `RRR Scarlet` (`Rz1`), and the same or greater resistance to
lodging as `RRR Scarlet` (`Rz1`). The expression of these traits may be
measured by a side by side phenotypic comparison, with differences and
similarities determined at a 5% significance level. Any such comparison
should be made in the same environmental conditions.

General Breeding and Selection Methods

[0073]Overview. Plant breeding is the genetic manipulation of plants. The
goal of wheat breeding is to develop new, unique and superior wheat
varieties. In practical application of a wheat breeding program, and as
discussed in more detail herein below, the breeder initially selects and
crosses two or more parental lines, followed by repeated `selfing` and
selection, producing many new genetic combinations. The breeder can
theoretically generate billions of different genetic combinations via
crossing, `selfing` and naturally induced mutations. The breeder has no
direct control at the cellular level, and two breeders will never,
therefore, develop exactly the same line. Each year, the plant breeder
selects the germplasm to advance to the next generation. This germplasm
may be grown under unique and different geographical, climatic and soil
conditions, and further selections may be made during and at the end of
the growing season.

[0074]Proper testing can detect major faults and establish the level of
superiority or improvement over current varieties. In addition to showing
superior performance, it is desirable that this a demand for a new
variety. The new variety should optimally be compatible with industry
standards, or create a new market. The introduction of a new variety may
incur additional costs to the seed producer, the grower, processor and
consumer, for special advertising and marketing, altered seed and
commercial production practices, and new product utilization. The testing
preceding release of a new variety should take into consideration
research and development costs as well as technical superiority of the
final variety. Ideally, it should also be feasible to produce seed easily
and economically.

[0075]These processes, which lead to the final step of marketing and
distribution, can take from six to twelve years from the time the first
cross is made. Therefore, development of new varieties is a
time-consuming process that requires precise forward planning, efficient
use of resources, and a focused direction. Various breeding and selection
methods are known in the art, and have substantial utility in the context
of particular aspects of the present invention.

[0076]Goals of Breeding. Wheat (Triticum aestivum L.), is an important and
valuable field crop, and a continuing goal of wheat breeders is to
develop stable, high yielding wheat varieties that are agronomically
sound and have good milling and baking qualities for its intended use. A
wheat breeder must therefore select and develop wheat plants that have
the traits that result in superior varieties. There are numerous steps in
the development of any novel and desirable plant germplasm. Plant
breeding begins with the analysis and definition of problems and
weaknesses of the current germplasm, the establishment of program goals,
and the definition of specific breeding objectives. This assessment is
followed by selection of germplasm that possess the traits to meet the
program goals; that is, to combine in a single variety an improved
combination of desirable traits from the parental germplasm. These
important traits may include, but are not limited to higher seed yield,
resistance to diseases and insects, tolerance to drought and heat,
improved grain quality, better agronomic qualities, herbicide resistance,
etc.

[0077]Breeding methods reflect pollination mode. Field crops are bred
through techniques that take advantage of the plant's method of
pollination. A plant is self-pollinated if pollen from one flower is
transferred to the same or another flower of the same plant. A plant is
sib-pollinated when individuals within the same family or line are used
for pollination. A plant is cross-pollinated if the pollen comes from a
flower on a different plant from a different family or line. The term
cross-pollination herein does not include self-pollination or
sib-pollination. Wheat plants (Triticum aestivum L.), are recognized to
be naturally self-pollinated plants which, while capable of undergoing
cross-pollination, rarely do so in nature. Thus, in the case of wheat,
intervention for control of pollination is critical to the establishment
of superior varieties. A cross between two different homozygous lines
produces a uniform population of hybrid plants that may be heterozygous
for many gene loci. A cross of two heterozygous plants each that differ
at a number of gene loci will produce a population of plants that differ
genetically and will not be uniform. Regardless of parentage, plants that
have been self-pollinated and selected for type for many generations
become homozygous at almost all gene loci and produce a uniform
population of true breeding progeny.

[0078]The term `homozygous plant` is hereby defined as a plant with
homozygous genes at 95% or more of its loci.

[0079]The term "inbred" as used herein refers to a homozygous plant or a
collection of homozygous plants.

[0080]Choice of breeding or selection methods. Choice of breeding or
selection methods depends on the mode of plant reproduction, the
heritability of the trait(s) being improved, and the type of variety used
commercially (e.g., F1 hybrid variety, pureline variety, etc.). For
highly heritable traits, a choice of superior individual plants evaluated
at a single location will be effective, whereas for traits with low
heritability, selection should be based on mean values obtained from
replicated evaluations of families of related plants. The complexity of
inheritance also influences choice of the breeding method. Breeding
generally starts with cross-hybridizing two genotypes (a "breeding
cross"), each of which may have one or more desirable characteristics
that is lacking in the other or which complements the other. If the two
original parents do not provide all the desired characteristics, other
sources can be included by making more crosses. In each successive filial
generation (e.g., F1→F2; F2→F3; F3 →F4;
F4→F5, etc.), plants are `selfed` to increase the homozygosity of
the line. Typically in a breeding program five or more generations of
selection and `selfing` are practiced to obtain a homozygous plant. Each
wheat breeding program should include a periodic, objective evaluation of
the efficiency of the breeding procedure. Evaluation criteria vary
depending on the goal and objectives, but should include gain from
selection per year based on comparisons to an appropriate standard,
overall value of the advanced breeding lines, and number of successful
varieties produced per unit of input (e.g., per year, per dollar
expended, etc.).

[0081]Promising advanced breeding lines are thoroughly tested and compared
to appropriate standards in environments representative of the commercial
target area(s), or Areas of Adaptability; that is, the location with the
environmental conditions that would be well suited for this wheat
variety. Area of adaptability is based on a number of factors, for
example: days to heading, winter hardiness, insect resistance, disease
resistance, and drought resistance. Area of adaptability does not
indicate that the wheat variety will grow in every location within the
area of adaptability or that it will not grow outside the area. Exemplary
areas of adaptability are: Northern area=States of DE, IL, IN, MI, MO,
NJ, NY, OH, PA, WI and Ontario, Canada; Mid-south=States of AR, KY, MO
and TN; Southeast=States of NC, SC, and VA; Deep South=States of AL, GA,
LA, and MS. The best lines are candidates for new commercial varieties;
those still deficient in a few traits may be used as parents to produce
new populations for further selection.

[0082]Identification of individuals that are genetically superior is a
difficult task because for most traits the true genotypic value is masked
by other confounding plant traits or environmental factors. One method of
identifying a superior genotype is to observe its performance relative to
other experimental genotypes and to a widely grown standard variety.
Generally, a single observation is inconclusive, so replicated
observations are required to provide a better estimate of its genetic
worth. A breeder uses various methods to help determine which plants
should be selected from the segregating populations and ultimately which
lines will be used for commercialization. In addition to the knowledge of
the germplasm and other skills used by a breeder, a part of the selection
process is dependent on experimental design coupled with the use of
statistical analysis. Experimental design and statistical analysis are
used to help determine which plants, which family of plants, and finally
which lines are significantly better or different for one or more traits
of interest. Experimental design methods are used to control error so
that differences between two lines can be more accurately determined.
Statistical analysis includes the calculation of mean values,
determination of the statistical significance of the sources of
variation, and the calculation of the appropriate variance components.
Five and one percent significance levels are customarily used to
determine whether a difference that occurs for a given trait is real or
due to the environment or experimental error.

[0084]Pedigree breeding. Pedigree breeding is commonly used for the
improvement of self-pollinating crops (e.g., wheat, etc). Two parents
that possess favorable, complementary traits are crossed to produce an
F1. An F2 population is produced by `selfing` or `sibbing` one or several
F1's. Selection of the best individuals may begin in the F2 population,
and beginning in the F3, the best individuals in the best families are
selected. Replicated testing of families can begin in the F4 generation
to improve the effectiveness of selection for traits with low
heritability. At an advanced stage of inbreeding (e.g., F5, F6 and F7),
the best lines or mixtures of phenotypically similar lines are tested for
potential release as new varieties.

[0085]Backcross breeding. Backcross breeding may be used to transfer genes
for simply inherited, qualitative, traits from a donor parent into a
desirable homozygous variety that is utilized as the recurrent parent.
The source of the traits to be transferred is called the donor parent.
After the initial cross, individuals possessing the desired trait or
traits of the donor parent are selected and then repeatedly crossed
(backcrossed) to the recurrent parent. The resulting plant is expected to
have the attributes of the recurrent parent (e.g., variety) plus the
desirable trait or traits transferred from the donor parent. This
approach has been used extensively for breeding disease-resistant
varieties.

[0086]Recurrent selection. Various recurrent selection techniques are used
to improve quantitatively inherited traits controlled by numerous genes.
The use of recurrent selection in self-pollinating crops depends on the
ease of pollination and the number of hybrid offspring recovered from
each successful cross. Recurrent selection can be used to improve
populations of either self- or cross-pollinated crops. A genetically
variable population of heterozygous individuals is either identified or
created by intercrossing several different parents. The best plants are
selected based on individual superiority, outstanding progeny, or
excellent combining ability. The selected plants are intercrossed to
produce a new population in which further cycles of selection are
continued. Plants from the populations can be selected and
self-pollinated to create new varieties.

[0087]Single-seed descent and modified single-seed descent. Another
breeding method is single-seed descent. This procedure in the strict
sense refers to planting a segregating population, harvesting a sample of
one seed per plant, and using the one-seed sample to plant the next
generation. When the population has been advanced from the F2 to the
desired level of inbreeding, the plants from which lines are derived will
each trace to different F2 individuals. The number of plants in a
population declines each generation due to failure of some seeds to
germinate or some plants to produce at least one seed. As a result, not
all of the F2 plants originally sampled in the population will be
represented by a progeny when generation advance is completed. In a
multiple-seed procedure, wheat breeders commonly harvest one or more
spikes (heads) from each plant in a population and thresh them together
to form a `bulk.` Part of the `bulk` is used to plant the next generation
and part is put in reserve. The procedure has been referred to as
modified single-seed descent. The multiple-seed procedure has been used
to save labor at harvest. It is considerably faster to thresh spikes with
a machine than to remove one seed from each by hand for the single-seed
procedure. The multiple-seed procedure also makes it possible to plant
the same number of seeds of a population each generation of inbreeding.
Enough seeds are harvested to make up for those plants that did not
germinate or produce seed.

[0088]Bulk breeding. Bulk breeding can also be used. In the bulk breeding
method, an F2 population is grown. The seed from the populations is
harvested in bulk and a sample of the seed is used to make a planting the
next season. This cycle can be repeated several times. In general, when
individual plants are expected to have a high degree of homozygosity,
individual plants are selected, tested, and increased for possible use as
a variety.

[0089]Determination of homozygotic stability, phenotypic stability,
heritability and identity, and the use of `Marker Assisted Selection`
(MAS).` There are many analytical methods available to determine the
homozygotic stability, phenotypic stability, heritability and identity of
the wheat varieties. The oldest and most traditional method of analysis
is the observation of phenotypic traits. The data is usually collected in
field experiments over the life of the wheat plants to be examined.
Phenotypic characteristics most often observed are for traits such as
seed yield, head configuration, glume configuration, seed configuration,
lodging resistance, disease resistance, maturity, etc.

[0090]In addition to phenotypic observations, the genotype of a plant can
also be examined through segregation analysis or the use of
biotechnology. There are many art-recognized, laboratory-based techniques
available for the analysis, comparison and characterization of plant
genotype, including but not limited to Gel Electrophoresis, Isozyme
Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs),
Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed
Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting
(DAF), Sequence Characterized Amplified Regions (SCARs), Amplified
Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs),
and Single Nucleotide Polymorphisms (SNPs). One use of molecular markers
is Quantitative Trait Loci (QTL) mapping. QTL mapping is the use of
markers, which are known to be closely linked to alleles that have
measurable effects on a quantitative trait. Selection in the breeding
process is based upon the accumulation of `markers` linked to the
positive-effecting alleles and/or the elimination of the markers linked
to the negative-effecting alleles from the plant's genome.

[0091]Molecular markers can also be used during the breeding process for
the selection of qualitative and quantitative traits. For example,
markers closely linked to alleles or markers containing sequences within
the actual alleles of interest can be used to select plants that contain
the alleles of interest during a backcrossing breeding program. The
markers can also be used to select for the genome of the recurrent parent
and against the markers of the donor parent. Using this procedure can
minimize the amount of genome from the donor parent that remains in the
selected plants. It can also be used to reduce the number of crosses back
to the recurrent parent needed in a backcrossing program (Openshaw et al.
Marker-assisted Selection in Backcross Breeding. In: Proceedings
Symposium of the Analysis of Molecular Marker Data, 5-6 Aug. 1994, pp.
41-43. Crop Science Society of America, Corvallis, Oreg.). The use of
molecular markers in the selection process is often called `Genetic
Marker Enhanced Selection` or `Marker Assisted Selection` (MAS).

[0092]Use of `double haploids` (DH). The production of `double haploids`
can also be used for the development of homozygous lines in a breeding
program. Double haploids are produced by the doubling of a set of
chromosomes (1N) from a heterozygous plant to produce a completely
homozygous individual. This can be advantageous because the process omits
the generations of `selfing` otherwise needed to obtain a homozygous
plant from a heterozygous source. Various methodologies of making double
haploid plants in wheat are known in the art (e.g., Laurie, D. A. and S.
Reymondie, Plant Breeding, 1991, v. 106:182-189; Singh, N. et al., Cereal
Research Communications, 2001, v. 29:289-296; Redha, A. et al., Plant
Cell Tissue and Organ Culture, 2000, v. 63:167-172; and U.S. Pat. No.
6,362,393).

[0093]Use of `hybrid` wheat. Though pure-line varieties are the
predominate form of wheat grown for commercial wheat production hybrid
wheat is also used. Hybrid wheat plants are produced with the help of
cytoplasmic male sterility, nuclear genetic male sterility, or chemicals.
Various combinations of these three male sterility systems have been used
in the production of hybrid wheat.

[0094]Tissue culture and regeneration. Further reproduction of the
root-rot-tolerant wheat genotypes of the invention can occur by tissue
culture and regeneration. Tissue culture of various tissues of wheat and
regeneration of plants therefrom is well known and widely published. A
review of various wheat tissue culture protocols can be found in "In
Vitro Culture of Wheat and Genetic Transformation-Retrospect and
Prospect" by Maheshwari et al. (Critical Reviews in Plant Sciences,
14(2): pp 149-178, 1995). Thus, another inventive aspect is to provide
cells that upon growth and differentiation produce wheat plants capable
of having the physiological and morphological characteristics of the
root-rot-tolerant wheat genotypes of the invention.

[0096]Also, contemplated by the instant invention are the nucleic acids
which comprise the genes which when expressed in the wheat plant, provide
root-rot resistance in wheat plants. The genetic sequences that comprise
mutations responsible for conferring root-rot tolerance to the wheat
plants of the present invention can be genetically mapped, identified,
isolated, and the sequence determined by those of ordinary skill in the
art (see e.g., Example 6 herein). See also, for example: Plant Genomes:
Methods for Genetic and Physical Mapping, J. S. Beckmann and T. C.
Osborn, 1992, Kluwer Academic Publishers; Genome Mapping in Plants,
Paterson, 1996, Harcourt Brace and Co.; Wheat Genome Mapping, A.
Kalinski, 1996, Diane Publishing Co.; and Methods in Molecular Biology,
Vol. 82, Arabidopsis Protocols, Martinez Zapater and Salinas, 1998,
Humana Press. Where the isolated nucleic acid encoding the genetic
element conferring the root-rot resistance encodes a protein responsible
for causing the plant to be root-rot resistant, the isolated nucleic acid
can be used to (1) identify other nucleic acids which may contain
mutations that provide root-rot resistance to wheat plants; (2) introduce
the isolated nucleic acid into a wheat plant which lacks root-rot
resistance by means of genetic engineering; (3) insert the isolated
nucleic acid into a suitable vector which can be expressed in a wheat
plant; and (4) insert the vector into a plant cell (e.g., a wheat plant
cell).

[0098]As used herein "operatively linked" refers to the linking of DNA
sequences (including the order of the sequences, the orientation of the
sequences, and the relative spacing of the various sequences) in such a
manner that the encoded protein is expressed. Methods of operatively
linking expression control sequences to coding sequences are well known
in the art. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y., 1982; and Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989.

[0099]"Expression control sequences" are DNA sequences involved in any way
in the control of transcription or translation. Suitable expression
control sequences and methods of making and using them are well known in
the art.

[0100]The expression control sequences preferably include a promoter. The
promoter may be inducible or constitutive. It may be naturally-occurring,
may be composed of portions of various naturally-occurring promoters, or
may be partially or totally synthetic. Guidance for the design of
promoters is provided by studies of promoter structure, such as that of
Harley and Reynolds, Nucleic Acids Res., 15, 2343-2361, 1987. Also, the
location of the promoter relative to the transcription start may be
optimized. See, e.g., Roberts et al., Proc. Natl. Acad. Sci. USA,
76:760-764, 1979.

[0102]Suitable inducible promoters for use in plants include: the promoter
from the ACE1 system which responds to copper (Mett et al., Proc. Natl.
Acad. Sci. 90:4567-4571, 1993): the promoter of the wheat In 2 gene which
responds to benzenesulfonomide herbicide safeners (U.S. Pat. No.
5,364,780 and Gatz et al., Mol. Gen. Genet. 243:32-38, 1994), and the
promoter of the Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genet.
227:229-237, 1991). According to one embodiment, the promoter for use in
plants is one that responds to an inducing agent to which plants normally
do not respond. An exemplary inducible promoter of this type is the
inducible promoter from a steroid hormone gene, the transcriptional
activity of which is induced by a glucosteroid hormone (Schena et al.,
Proc. Natl. Acad. Sci. 88:10421, 1991) or the application of a chimeric
transcription activator, XVE, for use in an estrogen receptor-based
inducible plant expression system activated by estradiol (Zou et al.,
Plant J. 24 265-273, 2000). Other inducible promoters for use in plants
are described in European Patent No. 332104, International Publication
No. WO 93/21334 and International Publication No. WO 97/06269, and
discussed in Gatz and Lenk Trends Plant Sci., 3:352-358, 1998, and Zou
and Chua, Curr. Opin. Biotechnol., 11:146-151, 2000. Finally, promoters
composed of portions of other promoters and partially or totally
synthetic promoters can be used. See, e.g., Ni et al., Plant J.
7:661-676, 1995, and International Publication No. WO 95/14098, which
describes such promoters for use in plants.

[0103]The promoter may include, or be modified to include, one or more
enhancer elements. Preferably, the promoter will include a plurality of
enhancer elements. Promoters containing enhancer elements provide for
higher levels of transcription as compared to promoters that do not
include them. Suitable enhancer elements for use in plants include the
PC1SV enhancer element (U.S. Pat. No. 5,850,019), the CaMV 35S enhancer
element (U.S. Pat. Nos. 5,106,739 and 5,164,316), and the FMV enhancer
element (Maiti et al., Transgenic Res., 6:143-156, 1997). See also,
International Publication No. WO 96/23898 and Enhancers and Eukaryotic
Expression (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1983).

[0104]For efficient expression, the coding sequences are preferably also
operatively linked to a 3' untranslated sequence. The 3' untranslated
sequence will preferably include a transcription termination sequence and
a polyadenylation sequence. The 3' untranslated region can be obtained
from the flanking regions of genes from Agrobacterium, plant viruses,
plants and other eukaryotes. Suitable 3' untranslated sequences for use
in plants include those of the cauliflower mosaic virus 35S gene, the
phaseolin seed storage protein gene, the pea ribulose-1,5-bisphosphate
carboxylase small subunit E9 gene, the wheat 7S storage protein gene, the
octopine synthase gene, and the nopaline synthase gene.

[0105]A 5' untranslated leader sequence can also be optionally employed.
The 5' untranslated leader sequence is the portion of an mRNA that
extends from the 5' CAP site to the translation initiation codon. This
region of the mRNA is necessary for translation initiation in plants and
plays a role in the regulation of gene expression. Suitable 5'
untranslated leader sequence for use in plants includes those of alfalfa
mosaic virus, cucumber mosaic virus coat protein gene, and tobacco mosaic
virus.

[0106]The DNA construct may be a `vector.` The vector may contain one or
more replication systems which allow it to replicate in host cells.
Self-replicating vectors include plasmids, cosmids and virus vectors.
Alternatively, the vector may be an integrating vector which allows the
integration into the host cell's chromosome of the DNA sequence encoding
the root-rot resistance gene product. The vector desirably also has
unique restriction sites for the insertion of DNA sequences. If a vector
does not have unique restriction sites it may be modified to introduce or
eliminate restriction sites to make it more suitable for further
manipulation.

[0107]Vectors suitable for use in expressing the nucleic acids, which when
expressed in a plant confer root-rot resistance, include but are not
limited to pMON979, pMON977, pMON886, pCaMVCN, and vectors derived from
the tumor inducing (Ti) plasmid of Agrobacterium tumefaciens described by
Rogers et al., Meth. Enzymol., 153:253-277, 1987. The nucleic acid is
inserted into the vector such that it is operably linked to a suitable
plant active promoter. Suitable plant active promoters for use with the
nucleic acids include, but are not limited to CaMV35S, ACTJN, FMV35S, NOS
and PCSLV promoters. The vectors comprising the nucleic acid can be
inserted into a plant cell using a variety of known methods. For example,
DNA transformation of plant cells include but are not limited to
Agrobacterium-mediated plant transformation, protoplast transformation,
electroporation, gene transfer into pollen, injection into reproductive
organs, injection into immature embryos and particle bombardment. These
methods are described more fully in U.S. Pat. No. 5,756,290, and in a
particularly efficient protocol for wheat described in U.S. Pat. No.
6,153,812, and the references cited therein. Site-specific recombination
systems can also be employed to reduce the copy number and random
integration of the nucleic acid into the cotton plant genome. For
example, the Cre/lox system can be used to immediate lox site-specific
recombination in plant cells. This method can be found at least in Choi
et al., Nuc. Acids Res. 28:B19, 2000).

Transgenes:

[0108]Molecular biological techniques allow the isolation and
characterization of genetic elements with specific functions, such as
encoding specific protein products. Scientists in the field of plant
biology developed a strong interest in engineering the genome of plants
to contain and express foreign genetic elements, or additional, or
modified versions of native or endogenous genetic elements in order to
alter the traits of a plant in a specific manner. Any DNA sequences,
whether from a different species or from the same species, that are
inserted into the genome using transformation are referred to herein
collectively as "transgenes." Several methods for producing transgenic
plants have been developed, and the present invention, in particular
embodiments, also relates to transformed versions of the
root-rot-tolerant wheat genotypes of the invention.

[0110]The most prevalent types of plant transformation involve the
construction of an expression vector. Such a vector comprises a DNA
sequence that contains a gene under the control of or operatively linked
to a regulatory element, for example a promoter. The vector may contain
one or more genes and one or more regulatory elements. Various genetic
elements can be introduced into the plant genome using transformation.
These elements include but are not limited to genes; coding sequences;
inducible, constitutive, and tissue specific promoters; enhancing
sequences; and signal and targeting sequences.

[0111]A genetic trait which has been engineered into a particular wheat
plant using transformation techniques could be moved into another line
using traditional breeding techniques that are well known in the plant
breeding arts. For example, a backcrossing approach could be used to move
a transgene from a transformed wheat plant to an elite wheat variety and
the resulting progeny would comprise a transgene. As used herein,
"crossing" can refer to a simple X by Y cross, or the process of
backcrossing, depending on the context. The term "breeding cross"
excludes the processes of selfing or sibbing.

[0112]With transgenic plants according to the present invention, a foreign
protein can be produced in commercial quantities. Thus, techniques for
the selection and propagation of transformed plants, which are well
understood in the art, yield a plurality of transgenic plants which are
harvested in a conventional manner, and a foreign protein then can be
extracted from a tissue of interest or from total biomass. Protein
extraction from plant biomass can be accomplished by known methods which
are discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-96,
1981.

[0113]According to a preferred embodiment, the transgenic plant provided
for commercial production of foreign protein is a wheat plant. In another
preferred embodiment, the biomass of interest is seed. A genetic map can
be generated, primarily via conventional RFLP, PCR, and SSR analysis,
which identifies the approximate chromosomal location of the integrated
DNA molecule. For exemplary methodologies in this regard, see Glick and
Thompson, Methods in Plant Molecular Biology and Biotechnology 269-284
(CRC Press, Boca Raton, 1993). Map information concerning chromosomal
location is useful for proprietary protection of a subject transgenic
plant. If unauthorized propagation is undertaken and crosses made with
other germplasm, the map of the integration region can be compared to
similar maps for suspect plants, to determine if the latter have a common
parentage with the subject plant. Map comparisons would involve
hybridizations, RFLP, PCR, SSR and sequencing, all of which are
conventional techniques.

Introduction of Transgenes of Agronomic Interest by Transformation

[0114]Agronomic genes can be expressed in transformed plants. For example,
plants can be genetically engineered to express various phenotypes of
agronomic interest, or, alternatively, transgenes can be introduced into
a plant by breeding with a plant that has the transgene. Through the
transformation of wheat, the expression of genes can be modulated to
enhance disease resistance, insect resistance, herbicide resistance,
water stress tolerance and agronomic traits as well as grain quality
traits. Transformation can also be used to insert DNA sequences which
control or help control male-sterility. DNA sequences native to wheat as
well as non-native DNA sequences can be transformed into wheat and used
to modulate levels of native or non-native proteins. Anti-sense
technology, various promoters, targeting sequences, enhancing sequences,
and other DNA sequences can be inserted into the wheat genome for the
purpose of modulating the expression of proteins. Exemplary genes
implicated in this regard include, but are not limited to, those
categorized below.

1. Genes that Confer Resistance to Pests or Disease:

[0115](A) Plant defenses are often activated by specific interaction
between the product of a disease resistance gene (R) in the plant and the
product of a corresponding avirulence (Avr) gene in the pathogen. A plant
variety can be transformed with a cloned resistance gene to engineer
plants that are resistant to specific pathogen strains. See, for example
Jones et al., Science 266: 789 (1994) (cloning of the tomato Cf-9 gene
for resistance to Cladosporium fulvum); Martin et al., Science 262: 1432
(1993) (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato
encodes a protein kinase); Mindrinos et al., Cell 78:1089, 1994
(Arabidopsis RSP2 gene for resistance to Pseudomonas syringae).

[0116]Fusarium head blight along with deoxynivalenol both produced by the
pathogen Fusarium graminearum Schwabe have caused devastating losses in
wheat production. Genes expressing proteins with antifungal action can be
used as transgenes to prevent Fusarium head blight. Various classes of
proteins have been identified. Examples include endochitinases,
exochitinases, glucanases, thionins, thaumatin-like proteins, osmotins,
ribosome inactivating proteins, flavoniods, lactoferricin. During
infection with Fusarium graminearum deoxynivalenol is produced. There is
evidence that production of deoxynivalenol increases the virulence of the
disease. Genes with properties for detoxification of deoxynivalenol (Adam
and Lemmens, In International Congress on Molecular Plant-Microbe
Interactions, 1996; McCormick et al. Appl. Environ. Micro. 65:5252-5256,
1999) have been engineered for use in wheat. A synthetic peptide that
competes with deoxynivalenol has been identified (Yuan et al., Appl.
Environ. Micro. 65:3279-3286, 1999). Changing the ribosomes of the host
so that they have reduced affinity for deoxynivalenol has also been used
to reduce the virulence of the Fusarium graminearum.

[0121](E) An insect-specific hormone or pheromone such as an ecdysteroid
and juvenile hormone, a variant thereof, a mimetic based thereon, or an
antagonist or agonist thereof. See, for example, the disclosure by
Hammock et al., Nature 344:458, 1990, of baculovirus expression of cloned
juvenile hormone esterase, an inactivator of juvenile hormone.

[0123](G) An enzyme responsible for an hyperaccumulation of a monterpene,
a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative
or another non-protein molecule with insecticidal activity.

[0124](H) An enzyme involved in the modification, including the
post-translational modification, of a biologically active molecule; for
example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a
nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a
phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a
chitinase and a glucanase, whether natural or synthetic. See PCT
application WO 93/02197 in the name of Scott et al., which discloses the
nucleotide sequence of a callase gene. DNA molecules which contain
chitinase-encoding sequences can be obtained, for example, from the ATCC
under Accession Nos. 39637 and 67152. See also Kramer et al., Insect
Biochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequence
of a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,
Plant Molec. Biol 21:673, 1993, providing the nucleotide sequence of the
parsley ubi4-2 polyubiquitin gene.

[0126](J) A hydrophobic moment peptide. See PCT application WO95/16776
(disclosure of peptide derivatives of Tachyplesin which inhibit fungal
plant pathogens) and PCT application WO95/18855 (teaches synthetic
antimicrobial peptides that confer disease resistance), the respective
contents of which are hereby incorporated by reference for this purpose.

[0127](K) A membrane permease, a channel former or a channel blocker. For
example, see the disclosure by Jaynes et al., Plant Sci. 89:43, 1993, of
heterologous expression of a cecropin-beta lytic peptide analog to render
transgenic tobacco plants resistant to Pseudomonas solanacearum.

[0131](O) A developmental-arrestive protein produced in nature by a
pathogen or a parasite. Thus, fungal endo alpha-1,4-D-polygalacturonases
facilitate fungal colonization and plant nutrient release by solubilizing
plant cell wall homo-alpha-1,4-D-galacturonase. See Lamb et al.,
Bio/Technology 10:1436, 1992. The cloning and characterization of a gene
which encodes a bean endopolygalacturonase-inhibiting protein is
described by Toubart et al., Plant J. 2:367, 1992.

[0132](P) A developmental-arrestive protein produced in nature by a plant.
For example, Logemann et al., Bio/Technology 10:305, 1992, have shown
that transgenic plants expressing the barley ribosome-inactivating gene
have an increased resistance to fungal disease.

[0140](B) A herbicide that inhibits the growing point or meristem, such as
an imidazalinone or a sulfonylurea. Exemplary genes in this category code
for mutant ALS and AHAS enzyme as described, for example, by Lee et al.,
EMBO J. 7: 1241, 1988, and Miki et al., Theor. Appl. Genet. 80: 449,
1990, respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659;
5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107;
5,928,937; and 5,378,824; and international publication WO 96/33270,
which are incorporated herein by reference for this purpose.

[0141](C) Glyphosate (tolerance, or resistance, imparted by mutant
5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,
respectively) and other phosphono compounds such as glufosinate
(phosphinothricin acetyl transferase, PAT) and Streptomyces hygroscopicus
phosphinothricin-acetyl transferase, bar, genes), and pyridinoxy or
phenoxy propionic acids and cycloshexones (ACCase inhibitor-encoding
genes). See, for example, U.S. Pat. No. 4,940,835 to Shah et al., which
discloses the nucleotide sequence of a form of EPSPS which can confer
glyphosate resistance. In U.S. Pat. No. 5,627,061 to Barry et al.
describes genes encoding EPSPS enzymes. In U.S. 2002/0062503 A1 Chen et
al. describe a wheat plant tolerant to glyphosate. The DNA construct
pMON30139 was inserted in wheat via transformation and contains the EPSPS
gene as well as other elements. See also U.S. Pat. Nos. 6,248,876 B1;
5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642;
4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060;
4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E; and U.S. Pat.
No. 5,491,288; and international publications WO 97/04103; WO 00/66746;
WO 01/66704; and WO 00/66747, which are incorporated herein by reference
for this purpose. Glyphosate resistance is also imparted to plants that
express a gene that encodes a glyphosate oxido-reductase enzyme as
described more fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, which are
incorporated herein by reference for this purpose. In addition glyphosate
resistance can be imparted to plants by the over expression of genes
encoding glyphosate N-acetyltransferase. See, for example, U.S.
Application Ser. Nos. 60/244,385; 60/377,175 and 60/377,719.

[0142]A DNA molecule encoding a mutant aroA gene can be obtained under
ATCC accession No. 39256, and the nucleotide sequence of the mutant gene
is disclosed in U.S. Pat. No. 4,769,061 to Comai. European Patent
application No. 0 333 033 to Kumada et al. and U.S. Pat. No. 4,975,374 to
Goodman et al. disclose nucleotide sequences of glutamine synthetase
genes which confer resistance to herbicides such as L-phosphinothricin.
The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is
provided in European application No. 0 242 246 to Leemans et al. De Greef
et al., Bio/Technology 7: 61, 1989, describe the production of transgenic
plants that express chimeric bar genes coding for phosphinothricin acetyl
transferase activity. See also, U.S. Pat. Nos. 5,969,213; 5,489,520;
5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024;
6,177,616 B1; and 5,879,903, which are incorporated herein by reference
for this purpose. Vasil et al. (Bio/Technology 10:667, 1992) reported
developing wheat plants resistant to glufosinate via particle bombardment
and the use of bar genes. The use of bar genes has also resulted in the
resistance to the herbicide bialaphos. Exemplary of genes conferring
resistance to phenoxy propionic acids and cycloshexones, such as
sethoxydim and haloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes
described by Marshall et al., Theor. Appl. Genet. 83:435, 1992.

[0143](D) A herbicide that inhibits photosynthesis, such as a triazine
(psbA and gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et
al., Plant Cell 3:169, 1991, describe the transformation of Chlamydomonas
with plasmids encoding mutant psbA genes. Nucleotide sequences for
nitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, and
DNA molecules containing these genes are available under ATCC Accession
Nos. 53435, 67441 and 67442. Cloning and expression of DNA coding for a
glutathione S-transferase is described by Hayes et al., Biochem. J.
285:173, 1992.

[0144](E) Protoporphyrinogen oxidase (protox) is necessary for the
production of chlorophyll, which is necessary for all plant survival. The
protox enzyme serves as the target for a variety of herbicidal compounds.
These herbicides also inhibit growth of all the different species of
plants present, causing their total destruction. The development of
plants containing altered protox activity which are resistant to these
herbicides are described in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1;
and 5,767,373; and international publication WO 01/12825, which are
incorporated herein by reference for this purpose.

[0147](C) Decreased phytate content for example introduction of a
phytase-encoding gene would enhance breakdown of phytate, adding more
free phosphate to the transformed plant. For example, see Van
Hartingsveldt et al., Gene 127:87, 1993, for a disclosure of the
nucleotide sequence of an Aspergillus niger phytase gene. See also U.S.
patent application Ser. Nos. 10/255,817 and 10/042,894 and international
publication numbers WO 99/05298, WO 03/027243, and WO 02/059324, which
are incorporated herein by reference for this purpose.

4. Genes that Control Male Sterility: [0149](A) Introduction of a
deacetylase gene under the control of a tapetum-specific promoter and
with the application of the chemical N-Ac-PPT (WO 01/29237). [0150](B)
Introduction of various stamen-specific promoters (WO 92/13956, WO
92/13957). [0151](C) Introduction of the barnase and the barstar gene
(Paul et al., Plant Mol. Biol. 19:611-622, 1992).5. Genes that Confer
Agronomic Enhancements, Nutritional Enhancements, or Industrial
Enhancements:

[0152](A) Improved tolerance to water stress from drought or high salt
water condition. The HVA1 protein belongs to the group 3 LEA proteins
that include other members such as wheat pMA2005, cotton D-7, carrot Dc3,
and rape pLEA76. These proteins are characterized by 11-mer tandem
repeats of amino acid domains which may form a probable amphophilic
alpha-helical structure that presents a hydrophilic surface with a
hydrophobic stripe. The barley HVA1 gene and the wheat pMA2005 gene are
highly similar at both the nucleotide level and predicted amino acid
level. These two monocot genes are closely related to the cotton D-7 gene
and carrot Dc3 gene with which they share a similar structural gene
organization. There is, therefore, a correlation between LEA gene
expression or LEA protein accumulation with stress tolerance in a number
of plants. For example, in severely dehydrated wheat seedlings, the
accumulation of high levels of group 3 LEA proteins was correlated with
tissue dehydration tolerance (Ried and Walker-Simmons, 1993). Studies on
several indica varieties of rice showed that the levels of group 2 LEA
proteins (also known as dehydrins) and group 3 LEA proteins in roots were
significantly higher in salt-tolerant varieties compared with sensitive
varieties. The barley HVA1 gene was transformed into wheat. Transformed
wheat plants showed increased tolerance to water stress, (Sivamani et al.
Plant Science 155:1-9, 2000, and U.S. Pat. No. 5,981,842).

[0153](B) Another example of improved water stress tolerance is through
increased mannitol levels via the bacterial mannitol-1-phosphate
dehydrogenase gene. To produce a plant with a genetic basis for coping
with water deficit, Tarczynski et al. (Proc. Natl. Acad. Sci. USA,
89:2600, 1992; WO 92/19731, published No. 12, 1992; Science 259:508,
1993) introduced the bacterial mannitol-1-phosphate dehydrogenase gene,
mtlD, into tobacco cells via Agrobacterium-mediated transformation. Root
and leaf tissues from transgenic plants regenerated from these
transformed tobacco cells contained up to 100 mM mannitol. Control plants
contained no detectable mannitol. To determine whether the transgenic
tobacco plants exhibited increased tolerance to water deficit, Tarczynski
et al. compared the growth of transgenic plants to that of untransformed
control plants in the presence of 250 mM NaCl. After 30 days of exposure
to 250 mM NaCl, transgenic plants had decreased weight loss and increased
height relative to their untransformed counterparts. The authors
concluded that the presence of mannitol in these transformed tobacco
plants contributed to water deficit tolerance at the cellular level. See
also U.S. Pat. No. 5,780,709 and international publication WO 92/19731
which are incorporated herein by reference for this purpose.

[0155]The transgenes described above can also be introduced into a
root-rot-tolerant plant of the present invention by conventional breeding
using as one parent a plant that has the transgene of interest.

Mutagenesis of Root-Rot-Tolerant Plants of the Invention

[0156]Further embodiments of the invention are the treatment of a root-rot
-tolerant wheat genotype of the invention with a mutagen and the plant
produced by such mutagenesis. Information about mutagens and mutagenizing
seeds or pollen are presented in the IAEA's Manual on Mutation Breeding
(IAEA, 1977) other information about mutation breeding in wheat can be
found in C. F. Konzak, "Mutations and Mutation Breeding" chapter 7B, of
Wheat and Wheat Improvement, 2nd edition, ed. Heyne, 1987.

Example 1

Methods for Chemical Mutagenesis of Zak, Scarlet, Macon, Hollis, Tara
2002, and Louise, and Screening of Same

[0157]Mutagenesis. General embodiments of the invention comprise the use
of the wheat cultivars for chemical mutagenesis. According to particular
aspects, the cultivars Zak and Scarlet (5,000 seeds each) were
mutagenized with ethane methyl sulfonate (EMS). Zak, Scarlet, Macon,
Hollis, Tara 2002, and Louise were mutagenized with ethane methyl
sulfonate (EMS; aka: ethyl methane sulfonate). Briefly, seeds were
presoaked in 200 ml 50 mM sodium phosphate buffer (Ph 7.0) for 5 hr, then
transferred to 200 ml of 0.3% EMS solution in phosphate buffer in a 2L
flask sealed and incubated with shaking for 16 hours at 22° C. An
equal volume of 10% sodium thiosulfate (w/v) was added to neutralize the
EMS and allowed to stand for 5 min before washing 10 times with water,
allowing the seeds to stand for 30 minutes in water between washes.

[0159]Screen for Rhizoctonia resistance. Mutagenized Zak and Scarlet are
evaluated for disease reaction to Rhizoctonia root rot as seedlings in a
growth chamber as described by Smith et al. 2003a. Briefly, two
treatments are used: 1) pasteurized soil (60° C. moist heat for 30
minutes) infested with ground oat grain inoculum; and, 2) pasteurized
soil only. Humidity is at 95% to limit plant transpiration and
evaporative water losses from the soil. Growth conditions can be 14-hour
day, with day and night temperatures of 23° C. and 11° C.,
respectively.

[0160]To grow Rhizoctonia, plastic tubes plugged with paper towel, are
filled with a layer of vermiculite to aid in aeration, followed by
soil/inoculum on top. Tubes are watered to near saturation and incubated
1 week to allow mycelium to colonize the soil. Two pre-germinated
mutagenized seeds are planted per tube. After 3 weeks, seedlings are
removed and scored for disease damage to roots.

Example 2

The Cultivars Zak and Scarlet were Chemically Mutagenized to Provide Novel
Wheat Genotypes Resistant to R. solani, R. oryzea and to Pythium; the Rz1
Genotype Segregated as a Single Gene, Semi-Dominant (Additive) Mutation

[0161]As described herein, Rhizoctonia resistance in wheat cultivars
(e.g., Zak or Scarlet) would revolutionize direct seeded spring wheat
production (e.g., in the Pacific North West PNW of the United States).
These resistant varieties also would serve as ideal gene donors for
future variety enhancement efforts in the PNW, across the United States
and worldwide, wherever soilborne disease pathogens like Rhizoctonia and
Pythium are problematic. Rhizoctonia solani AG-8 and R. oryzae cause
Rhizoctonia rot root of wheat and barley in the Pacific Northwest of the
United States and in cereal production regions throughout the world.
Acute (high) levels of R. solani AG-8 can cause bare patches in the
field, as the pathogen attacks young roots after they have emerged from
the seed. R. oryzae can cause Rhizoctonia damping-off, in which roots are
attacked during emergence from the seed. Therefore, in particular
aspects, the pathogen tolerance assays disclosed herein were conducted to
monitor both seedling tolerance to R. solani and/or R. oryzae, and
damping-off tolerance to R. oryzae.

[0162]Screening for Rhizoctonia root rot tolerance was performed in a wide
range of chemically mutagenized spring wheat cultivars produced. In years
one and two, 1085 and 1995 mutagenized plants from Zak and Scarlet,
respectively, were screened for tolerance to Rhizoctonia solani. From
this screening, a single mutant Scarlet line (015) (referred to herein as
`Rz1`, or as `RRR Scarlet`) has shown reproducible tolerance. Over the
past two years, 7,007 M2 lines of Macon, Hollis, Tara 2002 and Louise
have been screened for tolerance to R. solani, and four mutants from
Hollis and seven from Louise which may have tolerance to Rhizoctonia were
isolated.

[0163]The exemplary Rhizoctonia tolerant Scarlet line 015 has been
characterized in detail herein. Originally two Scarlet lines, 015 and
028, appeared to retest for Rhizoctonia root rot tolerance, but mutant
028 did not have strong tolerance so mutant 015 was characterized in most
detail. The presently disclosed results with 015 show that it gives
tolerance not only to R. solani, but also to R. oryzea and to Pythium.
Nonetheless, however, line 015 is highly susceptible to strip rust, a
foliar fungal pathogen, indicating that the mutation in 015 provides
tolerance to necrotrophic root pathogens, but not to pathogens that
infect the aerial part of the plant. This is a useful and validating
result, because mutations that are too general (e.g., that generally
impact resistance for many types of diseases) can have drawbacks that
adversely affect agronomic performance. Scarlet mutant 015 was grown in
the field in 2005, and was phenotypically indistinguishable from
unmutagenized Scarlet.

[0164]In particular aspects, three backcrosses of the Rhizoctonia tolerant
mutant to unmutagenized Scarlet (BC3 to Scarlet) were completed to
provide for germplasm release and to deploy the trait into adapted
germplasm (e.g., for use in gene deployment strategies).

[0166]a. The previously isolated Scarlet line 015 is used to introduce
good tolerance to Rhizoctonia solani, Rhizoctonia oryzae, and Pythium
into the spring wheat breeding program. The first step in this process is
to complete the three backcrosses to normal Scarlet required to "clean
up" the after-effects of mutagenesis. Genetic analysis of the first
backcross is complete. The second and third backcrosses have been made
and disease rating can be used to identify useful progeny for the
breeding program. Following the third backcross this gene can be deployed
throughout the spring wheat breeding program. [0167]b. The Rhizoctonia
tolerance gene can be mapped to develop a molecular marker linked to the
gene to speed deployment of the gene by reducing the amount of disease
evaluation needed to identify plants carrying the desired gene. [0168]c.
The degree of tolerance or resistance imparted by the gene from line 015
can be quantified using the real time PCR technique to measure the amount
of pathogen in the roots. [0169]d. Field testing can be used to determine
how well these plants perform in the field with and without disease
pressure using a Rhizoctonia nursery developed for in-field testing of
Rhizoctonia tolerance.

[0170]In additional aspects, the present applicants followed Rhizoctonia
seedling tolerance in two backcrosses of Scarlet Rz1 (BC1 and
BC2), and in two generations (F2 and F3 of BC1;
F3 and F4 of BC2) per backcross (for a total of different
four generations). The origin of Scarlet Rz1 populations used in the
tolerance assays was as follows:

##STR00001##

[0171]Rhizoctonia tolerance was found to be heritable in all generations
(FIGS. 4-8). The number of tolerant and susceptible individuals within
each group in each of the four generations was tracked to determine
whether tolerance was a single- or multi-gene trait (FIGS. 4-6).

[0172]To facilitate the assays, we used a combination of Rhizoctonia
solani AG-8 and R. oryzae (equal amounts of each pathogen). Pathogen
inoculum levels were adjusted to distinguish between tolerant and
susceptible individuals while achieving a moderate degree of infection,
that is, disease severity ratings of 4 to 6 for wild type Scarlet. It was
necessary to use higher pathogen levels (inoculum) for each successive
generation of Scarlet Rz1. Applicants postulate that the tolerance trait
was expressed more strongly in each generation as EMS-induced mutations
in other regions of the genome were eliminated by the process of gene
sorting or segregation. In general, the most reliable indicators of
pathogen tolerance were disease severity ratings of 0 to 1, high root
fresh weights and high total root lengths (FIG. 7). Shoot growth (FIG. 8)
and whole seedling weight (FIG. 6) also were positively correlated to
tolerance. BC2F4 plants derived from group 20-6R14 and
homozygous for Rhizoctonia tolerance were tested for seedling tolerance
to R. solani AG-8 (FIG. 9) or R. oryzae (FIG. 10). A separate experiment
to monitor damping-off tolerance to R. oryzae (FIG. 11) was conducted.
All BC2F4 plants of Scarlet Rz1 showed tolerance in all of the
assays.

[0173]Specifically, FIG. 4 shows, according to particular exemplary
embodiments of the present invention, frequency of seedling tolerance in
three generations of Scarlet Rz1, indicated by disease severity scores.
BC1F2 groups 20-2R and 21-SR (Column A) of Scarlet Rz1 were used to
derive BC1F3 groups (Column B). Second (BC2F3) backcross groups (Column
C) were derived from sibs of 20-2R and 21-5R. Plants were grown for 14
days in soil infested with R. solani AG-8 C1 plus R. oryzae 0801387.
BC1F2 assays were conducted using 200 ppg of each pathogen and 16
individuals per group; BC1F3 and BC2F3 assays used 250 ppg and 400 ppg of
each pathogen, respectively, and 24 individuals per group. Disease
severity was rated on a scale of 0 (no symptoms) to 8 (dead plant).
Groups were sorted into categories according to the highest ("resistant"
(R)), intermediate ("intermediate" (H)) or lowest ("susceptible" (S))
proportion of tolerant individuals within each group. The vertical dashed
lines indicate the mean disease severity score for each category.
Tolerance was not inherited in BC1F2 groups 23-3S and 24-1S, as all
individuals in these groups displayed disease ratings of 2 or greater.
However, tolerance in groups 20-2 and 20-6 was heritable in both
backcross generations, because at least some individuals displayed
disease ratings of 0 to 1.

[0174]FIG. 5 shows, according to particular exemplary embodiments of the
present invention, average root length values obtained from individuals
of the first (BC1F2) and second (BC2F3) backcross groups of Scarlet Rz1,
wild type Scarlet (Wt) and non-inoculated wild type Scarlet control
("cont"). Total root length was obtained from digitized scans of roots
using WinRHIZO 6.0 (Regent Instruments, Inc., Quebec, Canada). Letters
indicate significant (P<0.05) differences among the means, determined
using the least significant difference test (Statistix 8.1, Analytical
Software, Tallahassee, Fla.). The horizontal dashed lines indicate mean
values of all groups. Groups with means greater than that of Wt carried
the tolerance trait. Groups with means in the same statistical class as
"cont" (a) were comprised of more tolerant individuals than groups with
means in the same statistical class as Wt (c). Based on this distinction,
the former groups might be homozygous for the tolerance trait. Homozygous
candidates include 20-2R, 21-SR, 20-2H6, 20-6H13, and 20-6R14.

[0175]FIG. 6 shows, according to particular exemplary embodiments of the
present invention, comparison of distribution of seedling weights of
individuals from three BC2F3 groups of Scarlet Rz1. Wild type Scarlet
with (Wt) and without (cont) pathogen challenge were included as
controls. Conditions for pathogen assays are as described in FIG. 5. A
group that is homozygous for Rhizoctonia tolerance would consist of all
individuals having seedling weights above the dotted line. Seedling
weights within group 20-6R14 were clustered, very similar to those of
non-inoculated Scarlet (cont), and distinct from those of Wt, indicating
that 20-6R14 was homozygous for the tolerance trait. These data confirmed
the findings shown in FIG. 5, which identified 20-6R14 as a possible
homozygote. All progeny derived from 20-6R14 also will be homozygous for
the trait.

[0177]FIG. 8 shows, according to particular exemplary embodiments of the
present invention, comparison of shoot height in 14-day-old Scarlet wild
type (Wt), plants of BC2F3 group 20-6R14, and susceptible sibs of
20-6R14, all used for FIG. 7. Seedlings were germinated for 2 to 4 days
prior to sowing in pasteurized Spillman (Palouse Silt Loam) soil infested
with a combination of Rhizoctonia solani AG-8 C1 and R. oryzae 0801387
(400 propagules per gram soil of each isolate). Plants were grown in the
greenhouse at 15° C.±1° C. with 12 hours per day
supplemental lighting. The 12 hr per day refers to supplemental lighting
only; the "total" photoperiod was day length of natural light. Tolerance
in Scarlet Rz1 group 20-6R14 is indicated by a noticeable increase in
shoot length compared to sibs of 20-6R14 and to Wt. This increased shoot
length is comparable to uninoculated Wt control plants.

[0178]FIGS. 9A and B show, according to particular exemplary embodiments
of the present invention, seedling tolerance of group 20-6R14 to R.
solani AG-8 isolate C1. Twenty four individuals of the BC2F4 group
20-6R14 from Scarlet Rz1 or 20-2S9 (BC1F3 susceptible sib of Scarlet Rz1)
were grown in soil infested with 20, 100 and 400 ppg of the pathogen.
Twelve plants of wild type Scarlet (Wt) were included as a control.
Disease severity was rated on a scale of 0 (no symptoms) to 8 (dead
plant) after 14 days. Mean disease severity (FIG. 9A) was significantly
decreased and root weight (FIG. 9B) was significantly enhanced in Line
20-6R14 compared to Wt and Scarlet Rz1 susceptible sibs at all pathogen
levels. Letters indicate significant (P<0.05) differences among the
means, determined using the least significant difference test (Statistix
8.1, Analytical Software, Tallahassee, Fla.). Reduced disease severity
ratings and enhanced root weights in 20-6R14 indicate tolerance to levels
of R. solani AG-8 as high as 400 ppg (or about 6 to 10 times higher than
levels found in the field) is present in the BC2F4 generation of Scarlet
Rz1.

[0179]FIGS. 10A and B show, according to particular exemplary embodiments
of the present invention, seedling tolerance of 20-6R14 to R. oryzae
isolate 0801387. Twenty four individuals of the BC2F4 group 20-6R14 of
Scarlet Rz1 or 20-2S9 (BC1F3 susceptible sib) were grown in soil infested
with 20, 100 and 400 ppg of the pathogen. Twelve plants of wild type
Scarlet (Wt) were included as a control. Disease severity was rated on a
scale of 0 (no symptoms) to 8 (dead plant) after 14 days. Mean disease
severity (FIG. 10A) was decreased and root weight (FIG. 10B) was enhanced
in Line 20-6R14 compared to Wt and the Scarlet Rz1 susceptible sibs at
all inoculum levels. Letters indicate significant (P<0.05) differences
among the means, determined using the least significant difference test
(Statistix 8.1, Analytical Software, Tallahassee, Fla.). Reduced disease
severity ratings and enhanced root weights in 20-6R14 indicate tolerance
to levels of R. oryzae as high as 400 ppg (or about 6 to 10 times higher
than levels found in the field) is present in the BC2F4 generation of
Scarlet Rz1.

[0181]In summary, four generations of Scarlet Rz1 (see TABLE 3 below) were
tested for tolerance to Rhizoctonia solani AG-8 isolate C1 and R. oryzae
isolate 0801387. For seedling tolerance assays, plants were germinated on
moist filter paper in Petri plates for 2 to 4 days prior to sowing in
Rhizoctonia-infested soil. For damping-off tolerance assays, seeds were
sown directly into Rhizoctonia-infested soil. All assays were carried out
on plants individually grown in 6-inch plastic containers (Stuewe & Sons,
Corvallis, Oreg.) containing 70 g of pasteurized Spillman soil with or
without the pathogens. Plants were maintained at 15±1° C., with
12 h daily supplemental lighting (66 to 90 umol/m2/sec). Roots of
each plant were washed free of soil and rated for disease symptoms on a
scale of 0 (no symptoms) to 8 (dead plant). Shoot length and root fresh
weight data were obtained. Digital images of roots were generated using a
HP ScanJet 5370 (Hewlett Packard, Palo Alto, Calif.), and total root
length was determined using WinRHIZO 5.0 (Regent Instruments, Inc.,
Quebec, Canada). Individual plants showing good tolerance in the assays
were "rescued" by transplantation to pots of soil without pathogen, and
grown in the greenhouse for advancement to the next generation. In these
cases, seedlings were left intact and root weights could not be taken.

[0182]The disclosed findings indicate that Rhizoctonia tolerance is
conferred by a single co-dominant gene or locus. Applicants also
identified a BC2F3 group (20-6R14) in which all tested
individuals gave highly uniform responses to the pathogens (example in
FIG. 6). The trait for tolerance appeared to be homozygous in this group,
and is expected to be homozygous in all generations derived from this
group.

Example 3

The Rz1 Genotypes were also Demonstrated to be Pythium Tolerant

[0183]The following Example shows, according to exemplary aspects of the
present invention, that the Scarlet Rz1 genotypes were also demonstrated
to be Pythium tolerant.

Example Overview/Background:

[0184]Pythium root rot, caused by Pythium species, occurs in virtually all
wheat fields in Washington State (Cook and Veseth, 1991, Paulitz and
Adams, 1993), and this disease may be the most yield-limiting disease of
wheat in North America (Cook and Veseth, 1991). Pythium spp. cause a
decrease in root mass, which leads to poor nutrient uptake, resulting in
variable crop stands, decreased tiller numbers, varying maturity dates
and yield losses (Weller and Cook, 1986). Grain yields of wheat grown in
Pythium-free soil have been reported to be 15% to 25% higher than those
of wheat grown in Pythium infested soil (Cook and Haglund, 1991, Cook et
al. 1987, Cook et al., 1980, Hering et al. 1987, Weller and Cook, 1986).
If embryo damage is severe, seedlings often fail to emerge when infected
with Pythium (Fukui et al., 1994). Pythium root rot is prevalent in cool,
wet soils covered with crop debris (Cook and Haglund 1991, Cook et al.
1987, Cook and Veseth, 1991), which is typical of direct-seeded wheat
fields. An increased awareness of the environmental impacts of
traditional tillage practices, such as wind and water erosion, nutrient
leaching, and decreased soil organic matter, has caused many growers to
shift to direct-seeded wheat production (Pannkuk et al., 1987, Weller and
Cook, 1986). This shift in production practices provides Pythium species
with an optimal environment for infecting wheat crops (Cook 1992, Cook et
al. 1990). Chamswarng and Cook (1985) isolated and identified 10 Pythium
species from soils in eastern Washington that were pathogenic to wheat.
They found P. aristosporum, P. volutum, P. ultimum, P. sylvaticum
complex, and P. irregulare to be the most virulent of the isolates they
identified. Ingram and Cook (1990) assessed the pathogenicity of four
Pythium species on wheat, peas, lentils, and barley, and P. ultimum and
P. irregulare were the most virulent species to wheat, which agreed with
other reports (Chamswarng and Cook, 1985). In a recent study,
Higginbotham et al. (2003) detected differences between species and among
isolates within species of Pythium collected from wheat fields throughout
Eastern Washington (Paulitz and Adams, 2003). Pythium debaryanum isolate
90136 and P. ultimam isolate 90039 were the most virulent of the isolates
evaluated (Higginbotham et al. 2003), and may prove useful in future
disease screenings of Triticum germplasm where identifying genetic
resistance for highly virulent isolates is the goal.

[0185]Higginbotham et al. (2004a, 2004b) recently evaluated the level of
tolerance to Pythium root rot among a diverse set of wheat germplasm
collected from all major wheat production regions in the United States.
Pythium debaryanum isolate 90136 and P. ultimum isolate 90038, identified
as the most virulent Pythium isolates on wheat, were used to infest
pasteurized soil, which was seeded with wheat genotypes and placed in a
growth chamber maintained at a constant 16° C., 12 hr photoperiod
and ambient humidity (Higginbotham et al. 2004a). Length of the first
leaf and plant height measurements were recorded, and roots were
digitally scanned to create computer files that were analyzed using
WinRhizo software. Significant (P<0.05) differences in susceptibility
were detected among wheat genotypes in the presence of both Pythium
species, and a significant (P<0.0001) correlation between plant
stunting and root loss was detected (Higginbotham et al. 2004b). Based on
both shoot and root measurements, Caledonia, Chinese Spring, MN97695 and
OR942504 appear to be highly susceptible to Pythium root rot, whereas
genotypes KS93U161, OH708 and Sunco were the most tolerant to this
disease. Genotypes with high levels of tolerance may be useful gene
donors for cultivar improvement efforts.

[0202]Two groups of Scarlet Rz1 BC1F2, designated 20-6 and 21-3,
were sibs of groups 20-2R and 21-5R that were used in the Rhizoctonia
experiments described herein above. Furthermore, tolerant individuals
from groups 20-6 and 21-3 were used to produce the BC2F3 and
BC2F4 generations that displayed Rhizoctonia tolerance.

[0203]Seedling germination and growth conditions for Pythium tolerance
assays were essentially as described for that of Rhizoctonia spp.
Twenty-four seedlings of groups 20-6 and 21-3 were sown in pasteurized
Spillman (Palouse silt loam) soil infested with 1000 propagules per gram
(ppg) each of Pythium ultimum isolate 0900119 plus P. irregulare isolate
0900101. Both isolates are highly pathogenic to wheat and barley
(Higginbotham et al. 2004; Ingram and Cook 1990; Paulitz and Adams 2003).
Plants were harvested 14 days after growth in the Pythium-infested soil.
First leaf length (mm) and total root length (cm) were used to evaluate
tolerance. Total root length was determined using WinRHIZO 5.0 (Regent
Instruments, Inc., Quebec, Canada). Analysis of variance was done using
Statistix vers. 8.1 (Analytical Software, Tallahassee, Fla.).

Results for this Example:

[0204]Pythium damage is subtle, and roots cannot be rated for disease
symptoms as in the case of Rhizoctonia damage. However, the length of the
first true leaf is a reliable indicator of Pythium damage (Higginbotham
et al. 2004), as seedling roots that are attacked early by Pythium do not
support normal foliar growth. Root length also is impacted by Pythium
attack.

[0205]Tolerance to a combination of P. ultimum and P. irregulare grp I was
observed in Scarlet Rz1 BC1F2 groups 20-6 and 21-3. A
proportion of plants within a BC1F2 group are expected to be
heterozygous for pathogen tolerance and show an intermediate degree of
tolerance, whereas some plants will be homozygous for either tolerance
(strong tolerance) or susceptibility (no tolerance). Uninoculated wild
type Scarlet displayed leaf length values above 100 mm, whereas
Pythium-challenged wild type Scarlet generally showed leaf length values
below 100 mm. As expected for a BC1F2 group, the
BC1F2 plants showed a range of leaf length values (FIG. 12).
Mean leaf length values are shown in TABLE 4.

[0206]Specifically, FIG. 12 shows distribution of first leaf length (mm)
among individuals of Scarlet wild type (Wt) and Scarlet Rz1 BC1F2
populations P20-6 and P21-3. Seedlings were germinated for 2 to 4 days,
then grown for 14 days in pasteurized Spillman soil infested with 1000
propagules per gram (ppg) each of Pythium ultimum isolate 0900119 and
1000 ppg P. irregulare grp I isolate 0900101. Length of the first true
leaf was measured at 14 days. The dotted line is the mean leaf length
derived from all individuals, and delineates uninoculated wild type
Scarlet plants from inoculated plants that sustained Pythium damage. Many
plants within BC1F2 group 20-6 and several from group 21-3 had first leaf
length values that fell above the dotted line. The findings indicate that
these plants were tolerant to a combination of P. ultimum and P.
irregulare to an extent that they resembled uninoculated wild type
Scarlet plants. n=number of individuals screened.

[0207]The plants from each BC1F2 group were divided into three
arbitrary classes based on first leaf length (L): (R) or strong
tolerance, where L>100; (I) or intermediate tolerance, where
80≦L≦100; and (S) or susceptible, where 30<L<80.
Significant (P<0.05) differences of the means of each class were
observed (FIG. 13), indicating that tolerance to Pythium is segregating
among individuals of the BC1F2 groups, as expected.

[0208]Specifically, FIG. 13 shows that a proportion of plants within a
BC1F2 group are expected to be heterozygous for Pythium tolerance and
show an intermediate degree of tolerance, whereas some plants will be
homozygous for either tolerance (strong tolerance) or susceptibility (no
tolerance). Controls and growth conditions are as described in FIG. 12.
Plants in BC1F2 groups 20-6 and 21-3 were sorted into three arbitrary
classes based on first leaf length: (R) or strong tolerance, where
L>100; (I) or intermediate tolerance, where 80≦L≦100;
and (S) or susceptible, where 30<L<80. Letters above the bars
indicate significant (P<0.05) differences among the means of each
class were observed, indicating that tolerance to Pythium is segregating
among individuals of the BC1F2 groups, as expected. Without the sorting,
leaf length values of tolerant (R) plants would be averaged with
susceptible (S) plants, with no apparent overall tolerance.

[0209]Individual plants from the R (strong tolerance) and S (susceptible)
leaf length classes were picked at random for root length analysis. Total
root length values appeared to be segregating among individuals of 20-6
and 21-3, and those plants with longer leaf length also had longer roots.
Mean total root length values for all groups are given in TABLE 4. In the
R class, a small but significant (P<0.05) increase in root length was
observed in Pythium-challenged BC1F2 plants compared to
Pythium-challenged wild type Scarlet (FIG. 14). Pythium tolerance was
therefore indicated by enhancement of both foliar and root growth. A
small degree of root length enhancement in tolerant plants is expected
because other EMS mutations that reduce plant health are likely present
in the BC1F2 generation; these would be eliminated in subsequent
generations by gene or chromosome sorting.

[0210]Specifically, FIG. 14 shows, to validate the Pythium tolerance
indicated by leaf measurements, plants from the R (strong tolerance) and
S (susceptible) leaf length classes (see FIG. 13) were picked at random
for root length analysis. Controls were wild type Scarlet (Wt) with and
without inoculum. Letters indicate significant (P<0.05) differences
among the means (LSD). As with leaf length values, root length values of
tolerant (R) plants would be averaged with susceptible (S) plants, with
no apparent overall tolerance if the sorting was omitted. In the R class,
a small but significant (P<0.05) increase in root length was observed
in Pythium-challenged BC1F2 plants compared to Pythium-challenged wild
type Scarlet. A small degree of root length enhancement in tolerant
plants is expected because other EMS mutations that reduce plant health
are likely present in the BC1F2 generation; these would be eliminated in
subsequent generations by gene or chromosome sorting. The findings show
that Pythium tolerance in BC1F2 plants of Scarlet Rz1 is indicated by
enhanced foliar as well as root growth.

[0212]The Example describes isolation of drought tolerant wheat plants
based on increased sensitivity to the plant hormone ABA (abscisic acid)
during seed germination. The present applicants identified 19 independent
mutants with increased drought tolerance. These lines were backcrossed to
Chinese spring, and evaluated in a preliminary field trial at Spillman
Farm in 2005. Four of the most promising mutants were crossed to the
Rhizoctonia tolerant Scarlet mutant (see above) to provide for
introducing these genes into adapted spring wheat germplasm.

[0213]Rationale. Low precipitation levels limit yield potential of both
spring and winter wheat grown in Eastern Washington, and the development
of drought tolerant varieties could result in increased grain yields in
the Pacific Northwest (PNW), U.S.A.

[0214]Methods. The screen used to isolate drought tolerant wheat plants
was based on previous work in the model plant Arabidopsis showing that
plants with increased sensitivity to the plant hormone ABA during seed
germination tend to be tolerant to drought stress. ABA is both the seed
dormancy hormone and the drought tolerance hormone. ABA application
inhibits seed germination in a concentration-dependent manner. Applicants
screened for mutants that were unable to germinate on a concentration of
ABA that is normally too low to inhibit seed germination. These "ABA
hypersensitive" mutants are more sensitive to ABA in germination than
normal plant. ABA is the signal from the roots to the shoots to conserve
water as the soil dries. By making plants more sensitive to ABA, they are
forced to conserve water earlier than normal. In the case of era1 mutants
in Arabidopsis and in canola, this results in a high degree of drought
tolerance.

[0215]Results. Applicants have characterized the drought tolerance of ABA
hypersensitive mutants of Chinese spring, Scarlet, and Zak. The first
screening was performed in a `lab rat` wheat genotype called `Chinese
spring`. Of the 25 lines found to be ABA hypersensitive based on seed
germination, 4 appeared to be drought tolerant using transpiration rate
measurements. Based on segregation analysis of eight BC1F2
plants from backcross to normal Chinese spring, 4 mutations appeared to
be semi-dominant, 2 dominant, and 1 recessive. Four putative drought
tolerant plants have been crossed to the Rhizoctonia tolerant Scarlet
mutant to provide for mapping genes associated with these traits and to
deploy drought tolerance into adapted spring wheat germplasm. ABA
hypersensitive plants were grown to evaluate the effect of these
mutations on growth under dry conditions.

[0216]When the screen was repeated a total of 27 ABA hypersensitive
mutants were isolated in Scarlet and 4 in Zak. Drought tolerance tests
indicate that 5 of the 27 Scarlet and 1 of Zak lines show drought
tolerance. The first backcross of 4 Zak and 14 Scarlet ABA hypersensitive
lines has been completed.

[0218]Based on the genetic segregation data disclosed herein, the
Rhizoctonia root rot resistance in Rz1 is controlled by a single,
dominant gene. According to additional aspects, the following strategy
are used to deploy this gene into adapted spring wheat germplasm through
traditional cross-hybridization techniques (Allard 1999). Since
resistance is conferred by a single dominant gene, a 1 (homozygous
resistant):2 (heterozygous):1 (homozygous susceptible) segregation ratio
is expected among self-pollinated progeny from a heterozygous plant when
challenged with the pathogens.

Forward Breeding

[0219]An adapted line (susceptible) is cross-hybridized to a
BC2F4 homozygous derivative of Scarlet Rz1 (resistant) to
create novel genetic combinations containing the resistance gene from
Rz1. Seed is planted and resulting F1 hybrid plants are allowed to
self-pollinate and resulting F2 seed are harvested. Seed is planted and
resulting F2 plants re challenged with the pathogens to select
resistant plants for advancement. Twenty-five percent of the F2
progeny are expected to be homozygous resistant. These lines are
self-pollinated, resulting F3 seed is harvested, and a 40 g
sub-sample is used to establish a single F3 plot in the field.
Single heads from 100-150 F3 plants are threshed individually to
establish F4 head row families. Following selection for grain
appearance, plant height, and general adaptation, seed from 30-50 plants
within each selected head row are bulk harvested to obtain F5 seed
for early generation, end-use quality and disease response assessment.
Prior to end-use quality assessment, seedlings from each selected F5
bulk are assayed for resistance to Rhizoctonia and Pythium root rot in
controlled environment assays. Resistant lines are then evaluated for
end-use quality potential. Following selection for end-use quality,
F5 seed is used to establish single location field plots for initial
yield evaluations during the following crop year. F6 seed from high
yielding lines is subjected to small-scale milling and baking analyses.
Advanced lines (F7) with superior agronomic and end-use quality
potential are entered into preliminary yield trial evaluations where
detailed notes on field performance, including grain yields, test
weights, plant heights, heading dates, disease and insect resistance
ratings and various quality characteristics, are recorded. Superior lines
are selected and advanced to the next generation for field evaluation in
replicated trials at multiple locations. Advanced lines with variety
release potential are evaluated in the regional variety testing trials to
assess agronomic performance in diverse environments for at least 2
years, targeting areas prone to damage to soil-borne fungal root
pathogens. Experimental lines that equal or exceed agronomic and end-use
quality standards over multiple site/years with demonstrated resistance
to Rhizoctonia and/or Pythium root rots are released for commercial
production.

Backcross Breeding

[0220]In particular aspects, a backcross strategy is used to introgress
the resistance gene from Rz1 into agronomically superior adapted spring
wheat varieties. An adapted line (susceptible recurrent parent) is
cross-hybridized to a BC2F4 homozygous derivative of Scarlet
Rz1 (resistant donor parent). Seed is planted and pollen from resulting
F1 hybrid (heterozygous) plants is used to pollinate the recurrent
parent to produce BC1F1 seed. BC1F1 seed is planted
and resulting plants are screened for resistance to Rhizoctonia root rot.
A 1 (resistant (heterozygous)) to 1 (susceptible (homozygous))
segregation ratio is expected among BC1F1 progeny. Pollen from
resistant BC1F1 plants is used to pollinate recurrent parent
plants, to generate the BC2F1 plants. Again, a 1 (resistant
(heterozygous)) to 1 (susceptible (homozygous)) segregation ratio is
expected among BC2F1 progeny. The cycle is repeated to create
BC3F1 plants. BC3F1 plants are challenged by the
pathogens, and resistant lines are allowed to self-pollinate. Individuals
within resistant BC3F2 families are challenged with the
pathogens to identify lines that are homozygous for the resistance gene.
25% of the BC3F2 are expected to be families to be homozygous
resistant. BC3F3 seed from homozygous resistant lines is used
to establish single plot field trials Agronomic and end-use quality
potential is assessed as previously described. Allard, R. W. 1999.
Principles of Plant Breeding. Second Edition. John Wiley and Sons, Inc.,
New York, N.Y.

Example 6

Mapping the Rz1 Mutation; Marker Assisted Selection (MAS)

[0221]The Example describes, according to further embodiments, mapping of
the Rz1 mutation to provide for Marker Assisted Selection (MAS).

[0222]Rationale. Biotechnology has revolutionized plant breeding by
providing tools, such as molecular markers, which can be used in
Marker-Assisted Selection (MAS) strategies to rapidly incorporate
associated genes into improved varieties (Paterson et al. 1991). The
development of molecular markers associated with beneficial traits offers
an opportunity to assay genotypes during the breeding process to ensure
that essential genes are in fact present in selected individuals.
Molecular markers have been developed and used to introgress genes for
resistance to various diseases into a wide array of crops (Paterson et
al. 1991; Cenci et al. 1999; Naik et al. 1998; Paltridge et al. 1998;
Quint et al. 2002; Ramalingam et al. 2002). The success and efficiency of
using MAS depends upon how closely the marker is associated with the
target gene of interest, and the ease with which the marker can be
selected for among segregating progeny.

[0223]As described herein, traditional breeding approaches are used to
incorporate Rz1 into adapted germplasm. Unfortunately, selecting for this
trait is challenging because pathogen resistance assays are
time-consuming and labor-intensive. Carefully-controlled conditions are
required to conduct disease screening assays, and 3 weeks are required to
collect resistance response data from 120 plants. To circumvent these
problems, development of laboratory-based assays using DNA tags
associated with Rz1 for use in Marker-Assisted Selection (MAS) strategies
is desired, and such markers are useful in rapidly deploying this gene
into adapted wheat germplasm. Using MAS will increase the rate of gene
deployment by reducing the need to conduct disease evaluations in the
growth chamber at every stage of early generation advancement. The
presence of DNA tags associated with Rz1 can be monitored during the
advancement process, eliminating the need to verify the tolerance
response in the growth chamber until a manageable number of genotypes
carrying the molecular markers for Rz1 have been identified. In such
instances, disease response is confirmed through growth chamber analyses
prior to field testing. With the ability to conduct MAS for Rz1,
incorporation of the gene into fifth generation breeding material in an
18 month time span is possible.

Localization of Rz1 to a Chromosomal Segment:

[0224]The disease resistance in Scarlet-Rz1 is the result of a mutation
created through EMS mutagenesis, which is know to generate C to T nucleic
acid transitions and, less frequently, small chromosomal deletions.
According to particular aspects, mutations that result in the creation of
Rz1 results from a loss or change in function. Where Rz1 is the result of
loss of gene function, it is possible to localize the gene to a
chromosome location based on Rhizoctonia resistance reactions of wheat
deletion lines (Qi et al. 2003; Sears 1966). This approach reveals the
chromosomal region to focus on for fine-mapping efforts.

[0225]In particular aspects, Chinese Spring nullisomic-tetrasomic and
deletion lines are screened (Qi et al. 2003; Sears 1966) for resistance
to Rhizoctonia spp. to localize Rz1 to a chromosome segment. These lines
are comprised of a wheat plant in which one whole chromosome has been
lost and replaced with an extra copy of a homeologous chromosome. For
example, a nulli-tetrasomic line might lack chromosome 3A, but would
carry two copies of chromosome 3B. For purposes of illustration, if this
line was resistant to Rhizoctonia root rot, this would suggest that Rz1
is located on chromosome 3A.

[0226]Wheat deletion lines. Once the Rhizoctonia resistant phenotype is
localized to a chromosome, wheat deletion lines for that chromosome are
used to further delineate the position of Rz1 (Qi et al. 2003; Sears
1966). Deletion lines are missing different segments of the same
chromosome, and can be used to determine what segment of a chromosome a
particular gene is located on through a process called deletion mapping.
At least four individuals for each genetic stock are screened for
resistance to Rhizoctonia root rot.

[0227]Disease screening procedures. Rhizoctonia solani AG-8 is used to
determine the resistance reaction of genetic stocks (Paulitz et al.
2003). In particular aspects, pasteurized soil is infested with 1.5 g
ground oat grain inoculum per 1000 g of rolled soil. Un-inoculated soil
is also included as a control. Soil is placed in sterile containers with
a cotton ball in the bottom to prevent soil loss. Soil is moistened with
distilled water, and containers are be incubated in a growth room at
16° C. with a 14 hr day-length for 1 week to allow mycelium to
colonize the soil. A pre-germinated seedling is transferred to each
container. After 3 weeks, seedlings are removed, roots washed with a high
pressure water stream, and plants are rated for pathogen damage based on
plant height, severity of disease symptoms, percentage of infected
seminal roots, root weight, and quantitative variables analyzed in
WinRhizo using root scans to determine total root length, average root
diameter (indicating amount of lateral roots) and number of root tips.

Identification of Molecular Markers Associated with Rz1 in a Double
Haploid (DH) Population:

[0228]In additional aspects, molecular markers associated with Rz1 are
identified in a double haploid (DH) population developed from a cross
between Scarlet-Rz1 and its susceptible sibling Scarlet-S. In certain
aspects, EMS mutagenesis generates changes (e.g., polymorphisms) in the
DNA sequences flanking the Rz1 gene. Given the nature of EMS mutagenesis,
these changes are likely to involve single nucleotides; however, small
deletions also are possible. Such polymorphisms are expected to be
located on the same chromosome as Rz1, either within or tightly
associated with the gene itself. A molecular marker associated with such
a polymorphism provides facilitation for monitoring for the presence of
Rz1, since it represents a unique mutation event on the Rz1 chromosome,
which should be absent from other wheat cultivars.

[0229]In particular aspects parental lines have been screened with 1116
SSRs, and 46 (4.1%) were determined to be polymorphic. According to
particular aspects, these markers facilitate determining the genetic
linkage map location and identifying DNA markers associated with Rz1. The
46 polymorphic SSR markers are used to initiate mapping efforts in an
established double haploid mapping population.

[0230]Alternatively, parental lines are screened for polymorphisms using
restriction site polymorphisms (Sequence Tagged Site (STS)) markers, and
microarray-based Single Feature Polymorphisms (SFPs).

[0231]Screening for linked markers using conventional SSR and STS markers.
Screening for polymorphisms between Scarlet-Rz1 and its susceptible
sibling Scarlet-S is first performed using SSR and STS markers. Because
these markers are easy to use, they are good initial screening tools. Rz1
is mapped to its chromosomal location through genetic linkage analysis as
described by Anderson et al. (1992) to provide a molecular marker
associated with the gene for use in MAS (Anderson 2000). Initial
cross-hybridizations to create a segregating mapping population for DNA
marker evaluation and trait analysis are made between resistant
Scarlet-Rz1 and susceptible Scarlet-S. A mapping population of
approximately 200 DH individuals derived through microspore culture (see
section "c") is genotyped with polymorphic SSR markers (Stephenson et al.
1998). The mapping population is evaluated for disease responses to R.
solani and R. oryzae in the growth chamber as described by Smith et al.
2003a The populations are also evaluated in replicated field trials in
disease prone areas when adequate seed quantities are available. Trait
data is co-aligned with marker data using MAPMAKER version 3.0 (Lander et
al. 1987) to identify molecular markers associated with the resistance
gene. Alternatively, SFP polymorphisms are screened for by using
microarray analyses.

[0232]Microarray-based SFP markers. A polymorphism detected by a single
probe in an oligonucleotide array is called a Single Feature Polymorphism
(SFP), where a feature refers to a single probe in the array (West et al.
2006). Single nucleotide changes can lead to differences in signal
intensity during microarray hybridization. Microarray analysis allows
screening of over 100,000 probes to identify differences between genomic
DNA samples isolated from parents of the mapping population. Once a
polymorphism has been detected, it must be converted into a conventional
marker in order to efficiently screen a large mapping population. Two
venues are currently available for identifying polymorphisms using this
method, the Diversity Array Technology (DArT) service and the Wheat
Affymetrix Gene Chip (Akbari et al. 2006).

[0233]Generating the double haploid (DH) mapping population. Where a
sufficient number of polymorphisms is detected between Scarlet-Rz1 and
Scarlet-S, a DH mapping population is generated, derived from the cross
of these two parent lines using microspore culture. Microspores are
immature pollen and have the gametic number (n) of chromosomes. They can
be induced to divide and to form embryos or calli, which provide an
efficient source of double haploid plants. Double haploids are
genetically homozygous and produce pure breeding lines, which can be used
for linkage map construction. The protocol of Kasha et al. (2003) can be
used to establish the microspore culture technique, which involves: a)
collecting tillers of F1 plants at the mid-to late-uninucleate
stage; b) pre-treatment of 1 to 6 tillers under laminar flow hood in a
petridish (150 mm×15 mm) containing sterile 0.4M mannitol solution
followed by incubation in the dark at 4° C. for 7-10 days; c)
microspore isolation, which involves: 1) grinding pretreated spikes in
mannitol solution followed by separation of interphase (band of viable
embryogenic microspores) created by maltose/mannitol density gradient
centrifugation technique, purification of microspores and transferring
them to culture medium at an optimum cell density; and (2) transferring
resulting embryoides to regeneration medium to obtain green plantlets.
Green plantlets can be recovered in 5 to 6 weeks using this procedure.

[0234]Disease screening. All individuals in the DH mapping population are
screened for disease reaction to Rhizoctonia spp. as described above. At
least 4 individual plants per DH line are evaluated.

Fine-Mapping Rz1 using SSR Markers to Evaluate a DH Population Generated
from a Cross Between Scarlet-Rz1 and Chinese Spring:

[0235]In particular aspects, Chinese Spring was chosen as a mapping parent
because preliminary parental screening results indicated that a
significant portion of the markers evaluated were polymorphic between
Chinese Spring and Scarlet-Rz1, and because mapping results generated
using Chinese Spring as a parent would be expected to align with results
from the deletion mapping approach described. Substantial genetic and
genomic resources derived from Chinese Spring also are available to
assist in gene mapping and cloning. Where the chromosome or chromosome
region that contains Rz1 is identified, mapping efforts are focused on
SSR markers within the chromosome region known to contain Rz1. This
approach is used to accurately determine the position of Rz1 relative to
markers on currently published wheat genetic and physical maps. Knowing
the Rz1 map position is useful for determining what other genes or
markers of interest are located in that chromosomal region. Where a
marker linked to Rz1 is identified, this marker is mapped relative to
polymorphic SSRs to accelerate the mapping process.

[0236]Mapping relative to SSR markers. In particular aspects, Rz1 is
mapped relative to polymorphic SSR markers using DH lines derived from
the cross of the resistant parent Scarlet-Rz1 and the susceptible parent
Chinese Spring. A collection of at least 300 SSRs is used to screen the
parents for polymorphism detection. Mapping is performed as described
above.

[0237]Generating the DH mapping population. In certain aspects, a mapping
population consisting of 250 to 500 DH lines derived from the pollen of
50 to 100 F1 plants is used to develop this mapping population as
described above.

[0238]Disease screening. All individuals in the DH mapping populations are
screened for disease reaction to Rhizoctonia spp. as described above. At
least 4 individual plants per DH line are evaluated. Alternatively,
markers such as STS markers are used.

[0239]In particular aspects, therefore, the chromosomal location of the
Rhizoctonia root rot resistance gene Rz1 is determined, and molecular
markers closely associated with this trait are identified. These markers
are used to assay for the presence of Rz1 among segregating progeny
through MAS, which eliminates the need to conduct laborious growth
chamber screening assays to identify resistant genotypes among large
numbers of individuals from breeding populations in early stages of
advancement. In further aspects, the markers accelerate the rate of
development of Rhizoctonia root rot resistant wheat varieties targeted to
direct-seeded production conditions. In such aspects, the risk of crop
loss due to Rhizoctonia damage is eliminated, thereby increasing profit
potential, enabling more growers to take advantage of the environmental
advantages associated with direct-seeded spring wheat production.

[0272]The foregoing invention has been described in detail by way of
illustration and example for purposes of clarity and understanding.
However, it will be obvious that certain changes and modifications such
as single locus modifications and mutations, somoclonal variants, variant
individuals selected from large populations of the plants of the instant
variety and the like may be practiced within the scope of the invention.

[0273]All publications, patents and patent applications mentioned in the
specification are indicative of the level of those skilled in the art to
which this invention pertains. All such publications, patents and patent
applications are incorporated by reference herein for the purpose cited
to the same extent as if each was specifically and individually restated
herein.